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Fusarium wilt is one of main phytopathology attacking tomato (Solanum lycopersicum L.) plantations in Brazil. Plant rhizosphere and endophytic beneficial microorganism are well known as plant growth promoters and biocontrol agents. The present study aims to evaluate the potential of different Bacillus strains as biocontrol agent to Fusarium oxysporum f. sp. lycopersici Race 3 strains; and also as plant growth promoting bacteria on Solanum lycopersicum cv Perinha. Different in vitro and greenhouse experiments were carried out to evaluate the direct and indirect bacterial-fungus antagonism, and they inoculation effects on plant traits. In vitro direct, metabolites, and volatile antagonism analysis demonstrated that B. toyonensis BR 10491(FORT 02) presented a broad antagonism to all tested race 3 FOL strains while B. megaterium BR 10466 (FORT 12), B. aryabhattai BR 10494 (FORT 25), B. stratosphericus BR 10438 (FORT 29) and B. cereus BR 10493 (FORT 113.1) strains showed significant antagonistic activity for at least two applied methods. Greenhouse pot experiments demonstrated a significant BCA effect of FORT 113.1 and FORT 02 against FOL Race 3 Fus 1302 strain during different tomato development stages (seedling, vegetative, and reproductive). Bacillus cereus (FORT 113.1) showed significantly higher shoot and height fresh weight, Chlorophyll a and Chlorophyll b content, stomata conductance, water use efficiency, and also a lower xylem infection percentage during vegetative and reproductive stages. Antioxidant enzymatic components analysis demonstrated a synergic effect of Fusarium and Bacillus inoculation, leading to a higher superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activity. In conclusion, the results suggest that strain FORT113.1 could be considered as a good candidate for production of new biofungicide with high potential to augment the existing biocontrol strategies., Competing Interests: Declarations. Conflicts of interest: The authors declare that they have no conflict of interest., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

In sugarcane, sequences related to the genus Sphingomonas have been widely detected by microbiome studies. In this work, the presence of bacteria of this genus was confirmed using culture-dependent and independent techniques. A collection of thirty isolates was obtained using semispecific cultivation conditions, and a specific PCR assay was applied to help confirm the isolates as belonging to the genus. A series of laboratory evaluations were carried out to identify potential properties among the isolates in the collection, which consequently allowed the identification of some most promising isolates for the development of new agricultural bioinputs. In a separate analysis, the culture-independent fluorescence in situ hybridization (FISH) methodology was applied to demonstrate the natural occurrence of Sphingomonas in different organs and tissues of sugarcane. The results showed the presence of bacteria of the genus in the spaces between cells (apoplast) of the culm parenchyma, in vessels in the region of the leaf vein, on the adaxial surface of the leaf blade, and on the root surface, sometimes close to the base of root hairs, which suggests extensive colonization on the host plant. In summary, the present study corroborates previous metagenomic amplicon sequencing results that indicated a high occurrence of Sphingomonas associated with sugarcane. This is the first study that uses approaches other than amplicon sequencing to confirm the occurrence of the genus in sugarcane and, at the same time, demonstrates potentially beneficial activities to be explored by sugarcane cultivation., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

A group of Gram-negative plant-associated diazotrophic bacteria belonging to the genus Nitrospirillum was investigated, including both previously characterized and newly isolated strains from diverse regions and biomes, predominantly in Brazil. Phylogenetic analysis of 16S rRNA and recA genes revealed the formation of a distinct clade consisting of thirteen strains, separate from the formally recognized species N. amazonense (the closest species) and N. iridis. Comprehensive taxonomic analyses using the whole genomes of four strains (BR 11140 T  = AM 18 T  = Y-2 T  = DSM 2788 T  = ATCC 35120 T , BR 11142 T  = AM 14 T  = Y-1 T  = DSM 2787 T  = ATCC 35119 T , BR 11145 = CBAmC, and BR 12005) supported the division of these strains into two species: N. amazonense (BR 11142  T and BR 12005) and a newly proposed species (BR 11140  T and BR 11145), distinct from N. iridis. The phylogenomic analysis further confirmed the presence of the new Nitrospirillum species. Additionally, MALDI-TOF MS analysis of whole-cell mass spectra provided further evidence for the differentiation of the proposed Nitrospirillum species, separate from N. amazonense. Analysis of chemotaxonomy markers (i.e., genes involved in fatty acid synthesis, metabolism and elongation, phospholipid synthesis, and quinone synthesis) revealed that the new species highlights high similarity and evolutionary convergence with other Nitrospirillum species. This new species exhibited nitrogen fixation ability in vitro, it has similar NifHDK protein phylogeny position with the closest species, lacked denitrification capability, but possessed the nosZ gene, enabling N 2 O reduction, distinguishing it from the closest species. Despite being isolated from diverse geographic regions, soil types, and ecological niches, no significant phenotypic or physiological differences were observed between the proposed new species and N. amazonense. Based on these findings, a new species, Nitrospirillum viridazoti sp. nov., was classified, with the strain BR 11140 T (DSM 2788 T , ATCC 35120 T ) designated as the type strain., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

As agricultural practices become more sustainable, adopting more sustainable practices will become even more relevant. Searching for alternatives to chemical compounds has been the focus of numerous studies, and bacteriocins are tools with intrinsic biotechnological potential for controlling plant diseases. We continued to explore the biotechnological activity of the bacteriocin Gluconacin from Gluconacetobacter diazotrophicus , PAL5 strain, by investigating this protein's antagonism against important tomato phytopathogens and demonstrating its effectiveness in reducing bacterial spots caused by Xanthomonas perforans . In addition to this pathogen, the bacteriocin Gluconacin demonstrated bactericidal activity in vitro against Ralstonia solanacearum and Pseudomonas syringae pv. tomato , agents that cause bacterial wilt and bacterial spots, respectively. Bacterial spot control tests showed that Gluconacin reduced disease severity by more than 66%, highlighting the biotechnological value of this peptide in ecologically correct formulations.

Gluconacetobacter diazotrophicus has been the focus of several studies aiming to understand the mechanisms behind this endophytic diazotrophic bacterium. The present study is the first global analysis of the early transcriptional response of exponentially growing G. diazotrophicus to iron, an essential cofactor for many enzymes involved in various metabolic pathways. RNA-seq, targeted gene mutagenesis and computational motif discovery tools were used to define the G. diazotrophicus fur regulon. The data analysis showed that genes encoding functions related to iron homeostasis were significantly upregulated in response to iron limitations. Certain genes involved in secondary metabolism were overexpressed under iron-limited conditions. In contrast, it was observed that the expression of genes involved in Fe-S cluster biosynthesis, flagellar biosynthesis and type IV secretion systems were downregulated in an iron-depleted culture medium. Our results support a model that controls transcription in G. diazotrophicus by fur function. The G. diazotrophicus fur protein was able to complement an E. coli fur mutant. These results provide new insights into the effects of iron on the metabolism of G. diazotrophicus , as well as demonstrate the essentiality of this micronutrient for the main characteristics of plant growth promotion by G. diazotrophicus .

Sugarcane is an economically important crop that is used for the production of fuel ethanol. Diazotrophic bacteria have been isolated from sugarcane tissues, without causing visible plant anatomical changes or disease symptoms. These bacteria can be beneficial to the plant by promoting root growth and an increase in plant yield. Different rates of Biological Nitrogen Fixation (BNF) were observed in different genotypes. The aim of this work was to conduct a comprehensive molecular and physiological analysis of two model genotypes for contrasting BNF efficiency in order to unravel plant genes that are differentially regulated during a natural association with diazotrophic bacteria. A next-generation sequencing of RNA samples from the genotypes SP70-1143 (high-BNF) and Chunee (low-BNF) was performed. A differential transcriptome analysis showed that several pathways were differentially regulated among the two BNF-contrasting genotypes, including nitrogen metabolism, hormone regulation and bacteria recognition. Physiological analyses, such as nitrogenase and GS activity quantification, bacterial colonization, auxin response and root architecture evaluation, supported the transcriptome expression analyses. The differences observed between the genotypes may explain, at least in part, the differences in BNF contributions. Some of the identified genes might be involved in key regulatory processes for a beneficial association and could be further used as tools for obtaining more efficient BNF genotypes.

Bradyrhizobium spp. are well known to mediate biological nitrogen fixation (BNF) as microsymbionts inhabiting nodules on leguminous plants. However, they may also contribute to plant growth via free-living N 2 fixation (FLNF) in association with non-legumes. Notably, several Bradyrhizobium strains from sugarcane roots display FLNF activity. Among them, Bradyrhizobium sacchari is a legume symbiotic species, whereas strains AG48 and M12 are non-symbiotic. In the present study, a phylogenomic approach was applied to study peculiarities of these and other Bradyrhizobium strains with respect to N fixation (nif) gene content in order to reveal genetic features that enable FNLF in Bradyrhizobium spp. All FLNF strains carry an ancestral 'non-symbiotic' nif-gene cluster (NSC). B. sacchari also contains a second 'symbiotic' nif-gene cluster (SC), a characteristic observed in only three of 156 evaluated genomes. B. sacchari stood out and presented a high level of sequence divergence between individual nif-gene homologues and we discuss scenarios for the evolutionary origin of these clusters. The transcript level of NSC nifH gene increased during FLNF, when compared to symbiotic conditions. The data suggest that sugarcane roots harbor diverse Bradyrhizobium spp. that are genetically adapted to a dynamic environment where leguminous and non-leguminous host plants are alternately available., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Plant growth-promoting bacteria (PGPB) are bacteria that have mechanisms that facilitate plant growth in stress conditions such as drought. The objective of this study was to characterize bacterial strains isolated from bromeliads roots in ironstone outcrops (Urucum Residual Plateau, Mato Grosso do Sul, Brazil) for plant growth-promoting under drought conditions. Firstly, we screened isolates with the presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Then, all isolates were tested for tolerance to drought, exopolysaccharides (EPS) production, indole-3-acetic acid (IAA)-producing abilities, phosphate and zinc solubilization, production of catalase and hydrolytic enzymes (amylase, cellulase, and protease). Germination assay and a pot experiment with maize plants submitted to well-watered and drought conditions were performed with the strains most promising (VBN11 and VBE23). Briefly, Bacillus cereus VBE23 showed in vitro higher ACC deaminase activity (3.83 and 2.52 µmol α-KB mg -1  h -1 in non-drought and drought conditions, respectively), tolerance to drought, EPS production and other mechanisms of plant growth promotion: solubilization of phosphate and zinc, ammonia production, catalase activity and production of hydrolytic enzymes (amylase, cellulase, and protease). Inoculation of strain VBE23 in maize seeds submitted to drought conditions showed higher germination concerning uninoculated seeds and inoculated with VBN11. Also, the results indicated that the isolate VBE23 provided higher values of fresh and dry biomass compared to the control of uninoculated treatment and inoculated with VBN11 under drought conditions. This is the first report on the PGPB from ironstone outcrops of Urucum Residual Plateau, Mato Grosso do Sul, Brazil. Thus, this bacterial isolate could be used as a strategy for the facilitation of plant growth in drought environments.

Bacterial transcriptome profiling in the presence of plant fluids or extracts during microbial growth may provide relevant information on plant-bacteria interactions. Here, RNA sequencing (RNA-Seq) was used to determine the transcriptomic profile of Herbaspirillum seropedicae strain HRC54 at the early stages of response to sugarcane apoplastic fluid. Differentially expressed gene (DEG) analysis was performed using the DESeq2 and edgeR packages, followed by functional annotation using Blast2GO and gene ontology enrichment analysis using the COG and KEGG databases. After 2 h of sugarcane apoplastic fluid addition to the H. seropedicae HRC54 culture, respectively, 44 and 45 genes were upregulated and downregulated. These genes were enriched in bacterial metabolism (e.g., oxidoreductase and transferase), ABC transporters, motility, secretion systems, and signal transduction. RNA-Seq expression profiles of 12 genes identified in data analyses were verified by RT-qPCR. The results suggested that H. seropedicae HRC54 recognized sugarcane apoplastic fluid as the host signal, and some DEGs were closely involved at the early stages of the establishment of plant-bacteria interactions., Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-021-02848-y., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest in the publication., (© King Abdulaziz City for Science and Technology 2021.)

Bacteria of the genus Bacillus can colonize endophytically and benefit several crops including the control of some pest orders. In view of the benefits provided by these microorganisms and in order to find out an efficient biotechnological control for the giant borer, our interest in studying the microorganisms in symbiosis with sugarcane and the giant borer has arisen, since there is no efficient chemical or biological control method for this pest. Therefore, endophytic Bacillus strains were isolated from three sugarcane niches (apoplast fluid, central internode cylinder and roots) and also from the giant borer larvae living inside sugarcane varieties grown in the Northeast region of Brazil. The taxonomical characterization (16S rRNA) of 157 Gram-positive isolates showed that 138 strains belonged to the Bacillus genus. The most representative species were phylogenetically closely related to B. megaterium (11.5%) followed by B. safensis (10.8%), B. cereus (8.9%), B. oleronius (8.9%), B. amyloliquefaciens (7.0%), and B. pacificus (6.4%). BOX-PCR analyses showed very distinct band pattern profiles suggesting a great diversity of Bacillus species within the sugarcane niches and the digestive tract, while the B. cereus group remained very closely clustered in the dendrogram. According to XRE biomarker analysis, eleven strains (FORCN005, 007, 008, 011, 012, 014, 067, 076, 092, 093, and 135) correspond to B. thuringiensis species. Additional studies using conserved genes ( glp , gmk , pta , and tpi ) indicated that most of these strains were phylogenetically closely related to B. thuringiensis and may be considered different subspecies. In conclusion, this study suggests that the culturable Bacillus species are greatly diversified within the plant niches and showed Bacillus species in the digestive tract of the giant borer for the first time. These results open new perspectives to understand the role and functions played by these microorganisms in symbiosis with this pest and also the possibility of developing an efficient biological control method for the giant borer using strains identified as the B. thuringiensis species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rocha, Negrisoli Júnior, de Matos, Gitahy, Rossi, Vidal and Baldani.)

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., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Studies on the bacterial diversity associated with wild plants are rare, especially on those that grow in association with bromeliads. In the present study, we isolated and identified epiphytic and endophytic bacteria from the roots of the bromeliads Dyckia excelsa, Dyckia leptostachya and Deuterocohnia meziana occurring in the "cangas" in the Pantanal from Mato Grosso do Sul State, Brazil. The epiphytic bacteria were isolated from washed roots, while the endophytic bacteria were isolated from surface disinfested roots. Bacterial representatives corresponding to each BOX-PCR fingerprint, as well as those that did not result in amplicons, were selected for 16S rDNA gene sequence analysis. The BOX-PCR data showed intrageneric and intraspecific diversity and could discriminate strains and identify their phenotypic characteristics. The 16S rDNA gene sequence and phylogeny analysis showed a higher occurrence of strains belonging to the genus Bacillus than Mycobacterium and Brevibacterium, which were found in lower numbers. Species from the Bacillus genus are well known for their sporulation capacity and longer survival in arid locations, such as the "cangas". This study clearly showed that the bromeliad species represent a vast reservoir of bacterial community diversity, and the cultivable strains represent a new source for biotechnological prospecting.

Background: Inoculation with Gluconacetobacter diazotrophicus has shown to influence root development in red rice plants, and more recently, the induced systemic tolerance (IST) response to drought was also demonstrated. The goal of this study was to evaluate the inoculation effect of G. diazotrophicus strain Pal5 on the amelioration of drought stress and root development in red rice ( Oryza sativa L.)., Methods: The experimental treatments consist of red rice plants inoculated with and without strain Pal5 in presence and absence of water restriction. Physiological, biochemical, and molecular analyses of plant roots were carried out, along with measurements of growth and biochemical components., Results: The plants showed a positive response to the bacterial inoculation, with root growth promotion and induction of tolerance to drought. An increase in the root area and higher levels of osmoprotectant solutes were observed in roots. Bacterial inoculation increased the drought tolerance and positively regulated certain root development genes against the water deficit in plants., Conclusion: G. diazotrophicus Pal5 strain inoculation favored red rice plants by promoting various root growth and developmental mechanisms against drought stress, enabling root development and improving biochemical composition.

Nine bacterial strains were previously isolated in association with pinewood nematode (PWN) from wilted pine trees. They proved to be nematicidal in vitro, and one of the highest activities, with potential to control PWN, was showed by Serratia sp. M24T3. Its ecology in association with plants remains unclear. This study aimed to evaluate the ability of strain M24T3 to colonize the internal tissues of the model plant Arabidopsis thaliana using confocal microscopy. Plant growth-promoting bacteria (PGPB) functional traits were tested and retrieved in the genome of strain M24T3. In greenhouse conditions, the bacterial effects of all nematicidal strains were also evaluated, co-inoculated or not with Bradyrhizobium sp. 3267, on Vigna unguiculata fitness. Inoculation of strain M24T3 increased the number of A. thaliana lateral roots and the confocal analysis confirmed effective bacterial colonization in the plant. Strain M24T3 showed cellulolytic activity, siderophores production, phosphate and zinc solubilization ability, and indole acetic acid production independent of supplementation with L-tryptophan. In the genome of strain M24T3, genes involved in the interaction with the plants such as 1-aminocyclopropane-1-carboxylate (ACC) deaminase, chitinolytic activity, and quorum sensing were also detected. The genomic organization showed ACC deaminase and its leucine-responsive transcriptional regulator, and the activity of ACC deaminase was 594.6 nmol α-ketobutyrate μg protein -1  μl -1 . Strain M24T3 in co-inoculation with Bradyrhizobium sp. 3267 promoted the growth of V. unguiculata. In conclusion, this study demonstrated the ability of strain M24T3 to colonize other plants besides pine trees as an endophyte and displays PGPB traits that probably increased plant tolerance to stresses.

Analyses of the spatial localization and the functions of bacteria in host plant habitats through in situ identification by immunological and molecular genetic techniques combined with high resolving microscopic tools and 3D-image analysis contributed substantially to a better understanding of the functional interplay of the microbiota in plants. Among the molecular genetic methods, 16S-rRNA genes were of central importance to reconstruct the phylogeny of newly isolated bacteria and to localize them in situ . However, they usually do not allow resolution for phylogenetic affiliations below genus level. Especially, the separation of opportunistic human pathogens from plant beneficial strains, currently allocated to the same species, needs genome-based resolving techniques. Whole bacterial genome sequences allow to discriminate phylogenetically closely related strains. In addition, complete genome sequences enable strain-specific monitoring for biotechnologically relevant strains. In this mini-review we present high resolving approaches for analysis of the composition and key functions of plant microbiota, focusing on interactions of diazotrophic plant growth promoting bacteria, like Azospirillum brasilense , with non-legume host plants. Combining high resolving microscopic analyses with specific immunological detection methods and molecular genetic tools, including especially transcriptome analyses of both the bacterial and plant partners, enables new insights into key traits of beneficial bacteria-plant interactions in holobiontic systems.

The stalk apoplast fluid of sugarcane contains different sugars, organic acids and amino acids that may supply the demand for carbohydrates by endophytic bacteria including diazotrophs P. tropica (syn. B. tropica) strain Ppe8, isolated from sugarcane, is part of the bacterial consortium recommended as inoculant to sugarcane. However, little information has been accumulated regarding this plant-bacterium interaction considering that it colonizes internal sugarcane tissues. Here, we made use of the RNA-Seq transcriptomic analysis to study the influence of sugarcane stalk apoplast fluid on Ppe8 gene expression. The bacterium was grown in JMV liquid medium (100 ml), divided equally and then supplemented with 50 ml of fresh JMV medium or 50 ml of apoplast fluid extracted from sugarcane variety RB867515. Total RNA was extracted 2 hours later, the rRNAs were depleted and mRNAs used to construct libraries to sequence the fragments using Ion Torrent technology. The mapping and statistical analysis were carried out with CLC Genomics Workbench software. The RNA-seq data was validated by RT-qPCR using the reference genes fliP1, paaF, and groL. The data analysis showed that 544 genes were repressed and 153 genes were induced in the presence of apoplast fluid. Genes that induce plant defense responses, genes related to chemotaxis and movements were repressed in the presence of apoplast fluid, indicating that strain Ppe8 recognizes the apoplast fluid as a plant component. The expression of genes involved in bacterial metabolism was regulated (up and down), suggesting that the metabolism of strain Ppe8 is modulated by the apoplast fluid. These results suggest that Ppe8 alters its gene expression pattern in the presence of apoplast fluid mainly in order to use compounds present in the fluid as well as to avoid the induction of plant defense mechanisms. This is a pioneer study showing the role played by the sugarcane apoplast fluid on the global modulation of genes in P. tropica strain Ppe8., Competing Interests: The authors have declared that no competing interests exist.

Aims: This study aimed to explore the possibility of using the Gluconacin from Gluconacetobacter diazotrophicus strain PAL5 in the biological control of diverse sugarcane phytopathogenic bacteria., Methods and Results: An in silico analysis was first employed to determine the phylogenetic relationship between Gram-negative/positive bacteriocin producers and Gluconacin. The analysis showed that this trait is widespread among tested bacterial species and a well-conserved gene within the Acetobacteraceae family. The bacteriocin gene (GDI_0415) present in the genome of strain PAL5 was than cloned in pDEST™17 and expressed in Escherichia coli BL21-AI™. A bioassay showed growth inhibition of Xanthomonas albilineans by the recombinant bacteriocin. Subsequent bioassays indicated different levels of antagonistic activity against the majority of the sugarcane phytopathogenic bacteria (Xanthomonas axonopodis pv. vasculorum, Acidovorax avenae subsp. avenae, Pseudomonas syringae pv. syringae, Xanthomonas vasicola pv. vasculorum). In addition, the bacteriocin was also antagonistic to some beneficial bacterial strains belonging to G. diazotrophicus and endophytic Bacillus species, which also colonize sugarcane plants., Conclusions: The GDI_0415 gene, responsible for the production of Gluconacin, is well conserved within the Acetobacteraceae family and presented antagonistic activity against phytopathogenic and a few beneficial sugarcane bacteria., Significance and Impact of the Study: The production of a recombinant protein, named Gluconacin, opens new avenues for the agro-biotechnology application in agriculture, mainly with regard to the sugarcane crop., (© 2018 The Society for Applied Microbiology.)

Nitrospirillum amazonense is a nitrogen-fixing bacterium that shows potential to promote plant growth when inoculated into sugarcane and rice plants. This microorganism has been the subject of biochemical and genetic characterization to elucidate important functions related to host plant interaction and growth promotion, including the determination of draft genome sequences of two strains, Y2 and CBAmC, the second of which is the aim of the present study. CBAmC has been isolated from sugarcane (Saccharum spp.), and is currently used in a sugarcane consortium inoculant with four other nitrogen-fixing bacterial strains. The present paper describes a significant improvement in the genome sequence and assembly for the N. amazonense strain CBAmC, and determination for the first time of a complete genome sequence for this bacterial species, using PacBio technology. The analysis of the genomic data obtained allowed the discovery of genes coding for metabolic pathways and cellular structures that may be determinant for the success of the bacterial establishment and colonization into the host sugarcane plant, besides conferring important characteristics to the inoculant. These include genes for the use of sucrose and N-glycans, biosynthesis of autoinducer molecules, siderophore production and acquisition, auxin and polyamine biosynthesis, flagellum, σ-fimbriae, a variety of secretion systems, and a complete denitrification system. Concerning genes for nitrogenase and auxiliary proteins, it was possible to corroborate literature data that in N. amazonense these probably had originated from horizontal gene transfer, from bacteria of the Rhizobiales order. The complete genomic sequence of the CBAmC strain of N. amazonense revealed that the bacterium harbors four replicons, including three chromosomes and one chromid, a profile that coincides with that of other two strains, according to literature data, suggesting that as a replicon pattern for the species. Finally, results of phylogenomic analyses in this work support the recent reclassification of the species, separating it from the Azospirillum genus. More importantly, results of the present work shall guide subsequent studies on strain CBAmC as well as the development of a sugarcane inoculant.

Paraburkholderia tropica (syn Burkholderia tropica) are nitrogen-fixing bacteria commonly found in sugarcane. The Paraburkholderia tropica strain Ppe8 is part of the sugarcane inoculant consortium that has a beneficial effect on yield. Here, we report a draft genome sequence of this strain elucidating the mechanisms involved in its interaction mainly with Poaceae. A genome size of approximately 8.75Mb containing 7844 protein coding genes distributed in 526 subsystems was de novo assembled with ABySS and annotated by RAST. Genes related to the nitrogen fixation process, the secretion systems (I, II, III, IV, and VI), and related to a variety of metabolic traits, such as metabolism of carbohydrates, amino acids, vitamins, and proteins, were detected, suggesting a broad metabolic capacity and possible adaptation to plant association., (Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Cr(VI) is a highly toxic metal produced by anthropogenic activity which may impact the environment, affecting plants and animals. In plants, chromium both as Cr(III) or Cr(VI) can be absorbed by roots, is poorly translocated and affects negatively plant growth. Plants used in phytoremediation need to cope with chromium toxicity. This work aimed to evaluate strains of Ochrobactrum tritici and Nitrospirillum amazonense, resistant and modified in order to become chromate whole-cell biosensors, as plant-protectors enabling plants to withstand contaminated soils. In vitro tests were performed in three rice varieties and one maize variety. Initial evaluations of Cr(VI) toxicity to plants showed that plants had different sensitivities and BRS 6 CHUÍ rice variety was the most resistant. The metal affected plant growth and development, essentially in roots which were totally inhibited in rice varieties at 500 μM. This effect was plant-dependent. Modified N. amazonense proved to protect maize plants independently of the inoculation dose but O. tritici showed plant specificity and some toxicity when inoculated at high numbers, inhibiting rice development but not maize. Inoculants were directly responsible for growth improvements of specific plant varieties at 1.25 ppm Cr(VI), a concentration which corresponds to a weak soil contamination. Improvements were observed relatively to the Cr(VI)-treated controls, but also relative to the untreated controls, i.e., the benefits went beyond a simple neutralization of inhibition brought by Cr(VI) toxicity.

Quantitative reverse transcription PCR (RT-qPCR) is an important tool for evaluating gene expression. However, this technique requires that specific internal normalizing genes be identified for different experimental conditions. To date, no internal normalizing genes are available for validation of data analyses for Herbaspirillum rubrisubalbicans strain HCC103, an endophyte that is part of the sugarcane consortium inoculant. This work seeks to identify and evaluate suitable reference genes for gene expression studies in HCC103 grown until middle log phase in sugarcane juice obtained from four sugarcane varieties or media with three different carbon sources. The mRNA levels of five candidate genes (rpoA, gyrA, dnaG, recA and gmK) and seven target genes involved in carbon metabolism (acnA, fbp, galE, suhB, wcaA, ORF_0127.0101 and _0127.0123) were quantified by RT-qPCR. Analysis of expression stability of these genes was carried out using geNorm and Normfinder software. The results indicated that the HCC103 dnaG and gyrA genes are the most stable and showed adequate relative expression level changes among the different sugarcane juices. The highest expression level was seen for ORF_0127.0101, which encodes a sugar transporter, in juice from sugarcane variety RB867515 and glucose as the carbon source. The suhB gene, encoding SuhB inositol monophosphatase, had a higher relative expression level on 0.5% glucose, 100% sugarcane juice from variety RB867515 and 0.5% aconitate. Together the results suggest that dnaG and gyrA genes are suitable as reference genes for RT-qPCR analysis of strain HCC103 and that juice from different sugarcane varieties modulates the expression of key genes involved in carbon metabolism.

Members of the genus Bradyrhizobium are well-known as nitrogen-fixing microsymbionts of a wide variety of leguminous species, but they have also been found in different environments, notably as endophytes in non-legumes such as sugarcane. This study presents a detailed polyphasic characterization of four Bradyrhizobium strains (type strain BR 10280 T ), previously isolated from roots of sugarcane in Brazil. 16S rRNA sequence analysis, multilocus sequence analysis (MLSA) and analysis of the 16S-23S rRNA internal transcribed spacer showed that these strains form a novel clade close to, but different from B. huanghuaihaiense strain CCBAU 23303 T . Average nucleotide identity (ANI) analyses confirmed that BR 10280 T represents a novel species. Phylogenetic analysis based on nodC gene sequences also placed the strains close to CCBAU 23303 T , but different from this latter strain, the sugarcane strains did not nodulate soybean, although they effectively nodulated Vigna unguiculata, Cajanus cajan and Macroptilium atropurpureum. Physiological traits are in agreement with the placement of the strains in the genus Bradyrhizobium as a novel species for which the name Bradyrhizobium sacchari sp. nov. is proposed.

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.

Among the members of the genus Burkholderia, Burkholderia tropica has the ability to fix nitrogen and promote sugarcane plant growth as well as act as a biological control agent. There is little information about how this bacterium metabolizes carbohydrates as well as those carbon sources found in the sugarcane juice that accumulates in stems during plant growth. Reverse transcription quantitative PCR (RT-qPCR) can be used to evaluate changes in gene expression during bacterial growth on different carbon sources. Here we tested the expression of six reference genes, lpxC, gyrB, recA, rpoA, rpoB, and rpoD, when cells were grown with glucose, fructose, sucrose, mannitol, aconitic acid, and sugarcane juice as carbon sources. The lpxC, gyrB, and recA were selected as the most stable reference genes based on geNorm and NormFinder software analyses. Validation of these three reference genes during strain Ppe8 growth on the same carbon sources showed that genes involved in glycogen biosynthesis (glgA, glgB, glgC) and trehalose biosynthesis (treY and treZ) were highly expressed when Ppe8 was grown in aconitic acid relative to other carbon sources, while otsA expression (trehalose biosynthesis) was reduced with all carbon sources. In addition, the expression level of the ORF_6066 (gluconolactonase) gene was reduced on sugarcane juice. The results confirmed the stability of the three selected reference genes (lpxC, gyrB, and recA) during the RT-qPCR and also their robustness by evaluating the relative expression of genes involved in glycogen and trehalose biosynthesis when strain Ppe8 was grown on different carbon sources and sugarcane juice.

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 PAL5 T 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. ccc A, and rid A genes were reduced in the Gdiaa01 as compared to PAL5 T . 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 PAL5 T . In conclusion, our study demonstrated that G. diazotrophicus PAL5 T 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.

The RT-qPCR technique needs a validated set of reference genes for ensuring the consistency of the results from the gene expression. Expression stabilities for 9 genes from Herbaspirillum seropedicae, strain HRC54, grown with different carbon sources were calculated using geNorm and NormFinder, and the gene rpoA showed the best stability values., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Gluconacetobacter diazotrophicus is a nitrogen-fixing, endophytic bacterium that has the potential to promote plant growth and increase yield. Genetically modified strains might get more benefits to host plants, including through expression of useful proteins, such as Cry toxins from B. thuringiensis, or enzymes involved in phytohormone production, proteins with antagonistic activity for phytopathogens, or that improve nutrient utilization by the plant. For that, expression systems for G. diazotrophicus are needed, which requires active promoters fused to foreign (or innate) genes. This article describes the construction of a G. diazotrophicus PAL5 promoter library using a promoter-less lacZ-bearing vector, and the identification of six active promoters through β-galactosidase activity assays, sequencing and localization in the bacterial genome. The characterized promoters, which are located on distinct regions of the bacterial genome and encoding either sense or antisense transcripts, present variable expression strengths and might be used in the future for expressing useful proteins.

TonB-dependent receptors in concert with the TonB-ExbB-ExbD protein complex are responsible for the uptake of iron and substances such as vitamin B12 in several bacterial species. In this study, Tn5 mutagenesis of the sugarcane endophytic bacterium Gluconacetobacter diazotrophicus led to the isolation of a mutant with a single Tn5-insertion in the promoter region of a tonB gene ortholog. This mutant, named Gdiaa31, displayed a reduced growth rate and a lack of response to iron availability when compared to the wild-type strain PAL5(T). Several efforts to generate null-mutants for the tonB gene by insertional mutagenesis were without success. RT-qPCR analysis demonstrated reduced transcription of tonB in Gdiaa31 when compared to PAL5(T). tonB transcription was inhibited in the presence of Fe(3+) ions both in PAL5(T) and in Gdiaa31. In comparison with PAL5(T), Gdiaa31 also demonstrated decreased nitrogenase activity and biofilm formation capability, two iron-requiring physiological characteristics of G. diazotrophicus. Additionally, Gdiaa31 accumulated higher siderophore levels in culture supernatant. The genetic complementation of the Gdiaa31 strain with a plasmid that carried the tonB gene including its putative promoter region (pP(tonB)) restored nitrogenase activity and siderophore accumulation phenotypes. These results indicate that the TonB complex has a role in iron/siderophore transport and may be essential in the physiology of G. diazotrophicus.

Brazilian sugarcane has been shown to obtain part of its nitrogen via biological nitrogen fixation (BNF). Recent reports, based on the culture independent sequencing of bacterial nifH complementary DNA (cDNA) from sugarcane tissues, have suggested that members of the Bradyrhizobium genus could play a role in sugarcane-associated BNF. Here we report on the isolation of Bradyrhizobium spp. isolates and a few other species from roots of sugarcane cultivar RB867515 by two cultivation strategies: direct isolation on culture media and capture of Bradyrhizobium spp. using the promiscuous legume Vigna unguiculata as trap-plant. Both strategies permitted the isolation of genetically diverse Bradyrhizobium spp. isolates, as concluded from enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) fingerprinting and 16S ribosomal RNA, nifH and nodC sequence analyses. Several isolates presented nifH phylotypes highly similar to nifH cDNA phylotypes detected in field-grown sugarcane by a culture-independent approach. Four isolates obtained by direct plate cultivation were unable to nodulate V. unguiculata and, based on PCR analysis, lacked a nodC gene homologue. Acetylene reduction assay showed in vitro nitrogenase activity for some Bradyrhizobium spp. isolates, suggesting that these bacteria do not require a nodule environment for BNF. Therefore, this study brings further evidence that Bradyrhizobium spp. may play a role in sugarcane-associated BNF under field conditions., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: Burkholderia species play an important ecological role related to xenobiosis, the promotion of plant growth, the biocontrol of agricultural diseases, and symbiotic and non-symbiotic biological nitrogen fixation. Here, we highlight our study as providing the first complete genome of a symbiotic strain of B. phenoliruptrix, BR3459a (=CLA1), which was originally isolated in Brazil from nodules of Mimosa flocculosa and is effective in fixing nitrogen in association with this leguminous species., Results: Genomic comparisons with other pathogenic and non-pathogenic Burkholderia strains grouped B. phenoliruptrix BR3459a with plant-associated beneficial and environmental species, although it shares a high percentage of its gene repertoire with species of the B. cepacia complex (Bcc) and "pseudomallei" group. The genomic analyses showed that the bce genes involved in exopolysaccharide production are clustered together in the same genomic region, constituting part of the Group III cluster of non-pathogenic bacteria. Regarding environmental stresses, we highlight genes that might be relevant in responses to osmotic, heat, cold and general stresses. Furthermore, a number of particularly interesting genes involved in the machinery of the T1SS, T2SS, T3SS, T4ASS and T6SS secretion systems were identified. The xenobiotic properties of strain BR3459a were also investigated, and some enzymes involved in the degradation of styrene, nitrotoluene, dioxin, chlorocyclohexane, chlorobenzene and caprolactam were identified. The genomic analyses also revealed a large number of antibiotic-related genes, the most important of which were correlated with streptomycin and novobiocin. The symbiotic plasmid showed high sequence identity with the symbiotic plasmid of B. phymatum. Additionally, comparative analysis of 545 housekeeping genes among pathogenic and non-pathogenic Burkholderia species strongly supports the definition of a new genus for the second branch, which would include BR3459a., Conclusions: The analyses of B. phenoliruptrix BR3459a showed key property of fixing nitrogen that together with genes for high tolerance to environmental stresses might explain a successful strategy of symbiosis in the tropics. The strain also harbours interesting sets of genes with biotechnological potential. However, the resemblance of certain genes to those of pathogenic Burkholderia raise concerns about large-scale applications in agriculture or for bioremediation.

16S rRNA gene sequence analysis of eight strains (BR 3299(T), BR 3296, BR 10192, BR 10193, BR 10194, BR 10195, BR 10196 and BR 10197) isolated from nodules of cowpea collected from a semi-arid region of Brazil showed 97 % similarity to sequences of recently described rhizobial species of the genus Microvirga. Phylogenetic analyses of four housekeeping genes (gyrB, recA, dnaK and rpoB), DNA-DNA relatedness and AFLP further indicated that these strains belong to a novel species within the genus Microvirga. Our data support the hypothesis that genes related to nitrogen fixation were obtained via horizontal gene transfer, as sequences of nifH genes were very similar to those found in members of the genera Rhizobium and Mesorhizobium, which are not immediate relatives of the genus Microvirga, as shown by 16S rRNA gene sequence analysis. Phenotypic traits, such as host range and carbon utilization, differentiate the novel strains from the most closely related species, Microvirga lotononidis, Microvirga zambiensis and Microvirga lupini. Therefore, these symbiotic nitrogen-fixing bacteria are proposed to be representatives of a novel species, for which the name Microvirga vignae sp. nov. is suggested. The type strain is BR3299(T) ( = HAMBI 3457(T)).

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 β-glucose, 3-O-substituted units of β-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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The genus Burkholderia represents a challenge to the fields of taxonomy and phylogeny and, especially, to the understanding of the contrasting roles as either opportunistic pathogens or bacteria with biotechnological potential. Few genomes of nonpathogenic strains, especially of diazotrophic symbiotic bacteria, have been sequenced to improve understanding of the genus. Here, we contribute with the complete genome sequence of Burkholderia phenoliruptrix strain BR3459a (CLA1), an effective diazotrophic symbiont of the leguminous tree Mimosa flocculosa Burkart, which is endemic to South America.

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The genome of the endophytic diazotrophic bacterial species Gluconacetobacter diazotrophicus PAL5 (PAL5) revealed the presence of a gum gene cluster. In this study, the gumD gene homologue, which is predicted to be responsible for the first step in exopolysaccharide (EPS) production, was insertionally inactivated and the resultant mutant (MGD) was functionally studied. The mutant MGD presented normal growth and nitrogen (N(2)) fixation levels but did not produce EPS when grown on different carbon sources. MGD presented altered colony morphology on soft agar plates (0.3% agar) and was defective in biofilm formation on glass wool. Most interestingly, MGD was defective in rice root surface attachment and in root surface and endophytic colonization. Genetic complementation reverted all mutant phenotypes. Also, the addition of EPS purified from culture supernatants of the wild-type strain PAL5 to the mutant MGD was effective in partially restoring wild-type biofilm formation and plant colonization. These data provide strong evidence that the PAL5 gumD gene is involved in EPS biosynthesis and that EPS biosynthesis is required for biofilm formation and plant colonization. To our knowledge, this is the first report of a role of EPS in the endophytic colonization of graminaceous plants by a nitrogen-fixing bacterium.

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.

Aims: To evaluate the colonization process of sugarcane plantlets and hydroponically grown rice seedlings by Gluconacetobacter diazotrophicus strain PAL5 marked with the gusA and gfp reporter genes., Methods and Results: Sugarcane plantlets inoculated in vitro with PAL5 carrying the gfp::gusA plasmid pHRGFPGUS did not present green fluorescence, but beta-glucuronidase (GUS)-stained bacteria could be observed inside sugarcane roots. To complement this existing inoculation methodology for micropropagated sugarcane with a more rapid colonization assay, we employed hydroponically grown gnotobiotic rice seedlings to study PAL5-plant interaction. PAL5 could be isolated from the root surface (10(8) CFU g(-1)) and from surface-disinfected root and stem tissues (10(4) CFU g(-1)) of inoculated plants, suggesting that PAL5 colonized the internal plant tissues. Light microscopy confirmed the presence of bacteria inside the root tissue. After inoculation of rice plantlets with PAL5 marked with the gfp plasmid pHRGFPTC, bright green fluorescent bacteria could be seen colonizing the rice root surface, mainly at the sites of lateral root emergence, at root caps and on root hairs., Conclusion: The plasmids pHRGFPGUS and pHRGFPTC are valid tools to mark PAL5 and monitor the colonization of micropropagated sugarcane and hydroponic rice seedlings., Significance and Impact of the Study: These tools are of use to: (i) study PAL5 mutants affected in bacteria-plant interactions, (ii) monitor plant colonization in real time and (iii) distinguish PAL5 from other bacteria during the study of mixed inoculants.

Background: Gluconacetobacter 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., Results: Gluconacetobacter 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., Conclusion: The 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.

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

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.

Collectively, the species Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis represent microorganisms of high economic, medical, and biodefense importance. Although the genetic correlation and pathogenic characteristics have been extensively dissected, the ecological properties of these three species in their natural environments remain poorly understood. Thus, a tractable marker for detecting these bacteria under specific environmental and physiological conditions is a valuable tool. With this purpose, a plasmid (pAD43-25) carrying a functional gfp gene sequence (gfpmut3A) was introduced into the wild-type strain Bacillus thuringiensis subsp. kurstaki S76, which bears approximately 11 plasmids, allowing constitutive synthesis of green fluorescent protein (GFP) during vegetative growth (strain S76GFP+). Additionally, this vector was transferred to a plasmid-cured (Cry-) B. thuringiensis host. Bright green cells were detected by fluorescence microscopy in both recombinants by 2 h after inoculation in liquid medium and could be seen throughout the remaining cultivation time until complete sporulation was accomplished. For strain S76GFP+ protein profile and plasmid DNA analyses indicate, respectively, that this recombinant maintained Cry proteins expression and resident plasmid outline. Thus, in addition to the potential of strain S76GFP+ as a marker organism in bacteria--plant interaction studies, the production and stability of active GFPmut3a make this unique expression system a useful experimental model to study adaptive changes of host-plasmid as well as plasmid-plasmid relationships in a population of cells stressed by the production of a recombinant protein.

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.

Nitrogen-fixing bacteria have been isolated from sugarcane in an endophytic and beneficial interaction that promotes plant growth. In this work, for the first time, the involvement of ethylene signalling in this interaction was investigated by molecular characterizing members of this pathway in sugarcane. The expression pattern of a putative ethylene receptor (SCER1) and two putative ERF transcription factors (SCERF1 and SCERF2) show exclusive modulation in plants inoculated with the diazotrophic endophytes. The gene expression profile of SCER1, SCERF1, and SCERF2 is differentially regulated in sugarcane genotypes that can establish efficient or inefficient associations with diazotrophic micro-organisms, exhibiting high or low biological nitrogen fixation (BNF) rates, respectively. In addition, SCER1, SCERF1, and SCERF2 expression is different in response to interactions with pathogenic and beneficial micro-organisms. Taken together, that data suggest that SCER1, SCERF1, and SCERF2 might participate in specific ethylene signalling cascade(s) that can identify a beneficial endophytic association, modulating sugarcane responses toward the diazotrophic endophytes.

In a previous study, nitrogen-fixing isolates were recovered from the rhizosphere of maize and from surface-sterilized leaves of sugar cane cultivated in Rio de Janeiro, Brazil. On the basis of 16S rRNA gene sequence similarities, these isolates were identified as belonging to the genus Burkholderia, and whole-cell-protein profiles demonstrated that they are closely related to each other. In the present study, novel isolates were recovered from the roots of different sugar-cane varieties cultivated in diverse geographical regions of Brazil. Twenty-one nitrogen-fixing isolates were analysed using polyphasic taxonomy criteria, including DNA-DNA relatedness, 16S rRNA gene sequence similarities, fatty acid profiles, whole-cell-protein patterns and multilocus enzyme electrophoresis profiles, as well as morphological, physiological and biochemical characterization. The analysis confirmed that these isolates belong to a novel species within the genus Burkholderia, for which the name Burkholderia silvatlantica sp. nov. is proposed. The type strain, SRMrh-20(T) (=LMG 23149(T)=ATCC BAA-1244(T)), was isolated from the rhizosphere of maize var. Avantis A2345 cultivated in Seropédica, Rio de Janeiro.

Endophytic nitrogen-fixing bacteria have been isolated from graminaceous plants such as maize, rice, and sugarcane. They are thought to promote plant growth, not only by fixing nitrogen, but also by the production of plant hormones. The molecular mechanisms involved in this interaction are not yet clear. In this work, the identification of a receptor-like kinase (RLK), named SHR5, which may participate in signal transduction involved in the establishment of plant-endophytic bacteria interaction is described for the first time. SHR5 seems to be part of a novel subclass of RLKs present in a wide range of plant species. The expression of this gene is down-regulated in sugarcane plants associated exclusively with beneficial endophytic bacteria and is not a general response caused by micro-organisms or abiotic stress. In addition, more successful sugarcane-endophytic bacteria associations have a more pronounced decrease in SHR5 expression, suggesting that SHR5 mRNA levels in plant cells are inversely related to the efficiency of the association.

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.

In an ecological survey of nitrogen-fixing bacteria isolated from the rhizosphere and as endophytes of sugarcane, maize and teosinte plants in Brazil, Mexico and South Africa, a new phylogenetically homogeneous group of N(2)-fixing bacteria was identified within the genus Burkholderia. This polyphasic taxonomic study included microscopic and colony morphology, API 20NE tests and growth on different culture media at different pH and temperatures, as well as carbon source assimilation tests and whole-cell protein pattern analysis. Analysis of 16S rRNA gene sequences showed 99.2-99.9 % similarity within the novel species and 97.2 % similarity to the closest related species, Burkholderia sacchari. The novel species was composed of four distinct amplified 16S rDNA restriction analysis groups. The DNA-DNA reassociation values within the novel species were greater than 70 % and less than 42 % for the closest related species, B. sacchari. Based on these results and on many phenotypic characteristics, a novel N(2)-fixing species is proposed for the genus Burkholderia, Burkholderia tropica sp. nov., with the type strain Ppe8(T) (=ATCC BAA-831(T)=LMG 22274(T)=DSM 15359(T)). B. tropica was isolated from plants grown in geographical regions with climates ranging from temperate subhumid to hot humid.

Aims: To clone and sequence polymerase chain reaction (PCR)-amplified glnB and nifH genes of the nitrogen-fixing bacteria Burkholderia brasilensis strain M130, B. tropicalis strain PPe8 and B. kururiensis strain KP23., Methods and Results: The glnB and nifH gene fragments were amplified by PCR using universal degenerated primers. A very high percentage of similarity for the nifH (100%) and glnB (96%) genes was observed between strains M130 and KP23. A similarity of 100% for the nifH gene was also observed between strains M130 and PPe8. However, the identity for the glnB gene was 98% and the similarity 88%. The phylogenetic tree of the nifH gene showed a very high degree of similarity to the 16S rDNA gene., Conclusions: The nitrogen-fixing bacteria of the Burkholderia genus formed a cluster separated from the other species of the genus mainly when the nifH rather than the glnB gene was used to construct the phylogenetic tree., Significance and Impact of the Study: Knowledge of the nifH and glnB gene sequences of B. brasilensis, B. tropicalis and B. kururiensis will support new studies on the diversity of these diazotrophs in natural environments.

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.