Herbaspirillum Spp Research Articles

Efficacy of Nitrogen and Phosphorus Removal and Microbial Characterization of Combined A2O-MBBR Constructed Wetlands

A combined anaerobic–anoxic–oxic moving bed biofilm reactor (A2O-MBBR) constructed wetlands process was used to treat low carbon-to-nitrogen (C/N) simulated sewage. The results showed that the removal rates of chemical oxygen demand (COD), ammonia nitrogen (NH4+-N), total nitrogen (TN), and total phosphorus (TP) by this process were 94.06%, 94.40%, 67.11%, and 84.57%, respectively, and the concentrations of COD, NH4+-N, TN, and TP in the effluent were lower than the Class I-A standard of GB18918-2002. In the anoxic zone, NH4+-N had an inhibitory effect on phosphorus uptake via phosphorus-accumulating organisms (PAOs). The highest community diversity was observed in the anoxic zone sludge at 24 d. During the water-quality-shock loads stage, microbial community diversity decreased in a combined A2O-MBBR constructed wetlands reactor. At the phylum level, bacteria within the mature activated sludge were dominated by Proteobacteria, while Planctomycetes bacteria were the dominant species in the constructed wetlands. At the genus level, Tolumonas spp. were the dominant species in the 12 d and 24 d constructed wetlands and the anaerobic zone, with relative abundance percentages ranging from 20.24 to 33.91%. In the water-quality-shock loads stage, they were replaced by denitrifying bacteria such as Herbaspirillum spp. Unclassified_Burkholderiales was the dominant species in the constructed wetlands, with a relative abundance of 33.09%.

Structural and genetic characterization of the colitose-containing O-specific polysaccharide from the lipopolysaccharide of Herbaspirillum frisingense GSF30T

The lipopolysaccharide (LPS) of Herbaspirillum frisingense GSF30T (HfGSF30), a non-pathogenic diazotrophic endobiont, was isolated by phenol-water extraction from bacterial cells and was characterized by chemical analyses and SDS PAGE. The O-specific polysaccharide (OPS, O-antigen), obtained by mild acid hydrolysis of the LPS, was examined by sugar and methylation analysis, along with 1H and 13C NMR spectroscopy, including 2D 1H,1H COSY, 1H,1H TOCSY, 1H,1H ROESY, 1H,13C HSQC, and 1H,13C HMBC experiments. The OPS was found to consist of branched tetrasaccharide repeating units of the following structure:→3)−α−L−Rhap−1→3−β−D−Galp−1→3−β−D−GlcpNAc−(1→2↑1α−ColpThis structure is unique among the known bacterial polysaccharide structures. Analysis of the HfGSF30 genome showed that it contained a set of sequentially arranged operons (presumably a cluster of genes) associated with the O-antigen. Amino acid sequence analysis using the BLAST program demonstrated the specificity of this putative cluster for Herbaspirillum spp. The genes responsible for the biosynthesis of the OPS of HfGSF30 were dispersed in the genome, constituting small operons. A putative O-antigen gene cluster of HfGSF30 was identified and found to be consistent with the OPS structure.

Bacterial Small RNAs in the Genus Herbaspirillum spp.

The genus Herbaspirillum includes several strains isolated from different grasses. The identification of non-coding RNAs (ncRNAs) in the genus Herbaspirillum is an important stage studying the interaction of these molecules and the way they modulate physiological responses of different mechanisms, through RNA–RNA interaction or RNA–protein interaction. This interaction with their target occurs through the perfect pairing of short sequences (cis-encoded ncRNAs) or by the partial pairing of short sequences (trans-encoded ncRNAs). However, the companion Hfq can stabilize interactions in the trans-acting class. In addition, there are Riboswitches, located at the 5′ end of mRNA and less often at the 3′ end, which respond to environmental signals, high temperatures, or small binder molecules. Recently, CRISPR (clustered regularly interspaced palindromic repeats), in prokaryotes, have been described that consist of serial repeats of base sequences (spacer DNA) resulting from a previous exposure to exogenous plasmids or bacteriophages. We identified 285 ncRNAs in Herbaspirillum seropedicae (H. seropedicae) SmR1, expressed in different experimental conditions of RNA-seq material, classified as cis-encoded ncRNAs or trans-encoded ncRNAs and detected RNA riboswitch domains and CRISPR sequences. The results provide a better understanding of the participation of this type of RNA in the regulation of the metabolism of bacteria of the genus Herbaspirillum spp.

Effect of Endophytic Bacterium Inoculation on Seed Germination and Sprout Growth of Tartary Buckwheat

This experiment was conducted to investigate the endophytic bacterium Herbaspirillum spp effect on seed germination and sprout growth of tartary buckwheat. Inoculant concentration (%v/v) and seed soaking time were applied 10, 20 and 40% and 0, 4, 8, 12 hour, respectively. The experiment was carried out in a growth chamber maintained temperature at 20, 25 and 30°C without light for 7 days. Results showed that, 10 to 20% (v/v) inoculant concentration by 4 to 8 h seed soaking time at 20°C temperature increased seed vigor rate and total seed germination rate 80-95% and 90-100%, respectively. On the other and, seed inoculation with Herbaspirillum spp. increased hypocotyl length (13-15 ㎝), root length (8-11 ㎝), total fresh weight (135-296 g) and total dry weight (7-10 g), compared to control. It is indicated that sprouts growth and yield depends on inoculation concentrations, seed soaking time and temperature. Therefore, it would be suggested that seed inoculation with Herbaspirillum spp. at concentration of 10 to 20% (v/v), soaking time 4 to 8 h and temperature 20°C promote seed germinations and sprout growth rate of tartary buckwheat.

Biochemical characteristics, adhesion, and cytotoxicity of environmental and clinical isolates of Herbaspirillum spp.

Herbaspirillum bacteria are best known as plant growth-promoting rhizobacteria but have also been recovered from clinical samples. Here, biochemical tests, matrix-assisted laser deionization-time of flight (MALDI-TOF) mass spectrometry, adherence, and cytotoxicity to eukaryotic cells were used to compare clinical and environmental isolates of Herbaspirillum spp. Discrete biochemical differences were observed between human and environmental strains. All strains adhered to HeLa cells at low densities, and cytotoxic effects were discrete, supporting the view that Herbaspirillum bacteria are opportunists with low virulence potential.

Quantification of natural populations of Gluconacetobacter diazotrophicus and Herbaspirillum spp. In sugar cane (Saccharum spp.) Using differente polyclonal antibodies

The species Gluconacetobacterdiazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans are endophytic N2-fixing [diazotrophic] bacteria which colonise not only roots, but also the aerial tissue of sugar cane. However, the technique most commonly used to quantify the populations of these microbes in plants is by culturing serial dilutions of macerates of plant tissues in N free semi-solid media which are only semi-selective for the species/genera [the Most Probable Number (MPN) Technique] and each culture must be further subjected to several tests to identify the isolates at the species level. The use of species-specific polyclonal antibodies with the indirect ELISA (enzyme-linked immunosorbent assay) can be an alternative which is rapid and specific to quantify these populations of bacteria. This study was performed to investigate the viability of adapting the indirect ELISA technique to quantify individually the populations of these three species of diazotroph within the root and shoot tissues of sugarcane. The results showed that species-specific polyclonal antibodies could be obtained by purifying sera in protein-A columns which removed non-specific immuno-globulins. It was possible to quantify the three bacterial species in the Brazilian sugarcane variety SP 70-1143 in numbers above 10(5) cells per g fresh weight in roots, rhizomes and leaves. The numbers of the different bacterial species evaluated using the ELISA technique were found to be higher than when the same populations were evaluated using the MPN technique, reaching 1400 times greater for G. diazotrophicus and 225 times greater for Herbaspirillum spp. These results constitute the first quantification of Herbaspirillum using immunological techniques.

Quantification of natural populations of Gluconacetobacter diazotrophicus and Herbaspirillum spp. In sugar cane (Saccharum spp.) Using differente polyclonal antibodies.

The species Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans are endophytic N2-fixing [diazotrophic] bacteria which colonise not only roots, but also the aerial tissue of sugar cane. However, the technique most commonly used to quantify the populations of these microbes in plants is by culturing serial dilutions of macerates of plant tissues in N free semi-solid media which are only semi-selective for the species/genera [the Most Probable Number (MPN) Technique] and each culture must be further subjected to several tests to identify the isolates at the species level. The use of species-specific polyclonal antibodies with the indirect ELISA (enzyme-linked immunosorbent assay) can be an alternative which is rapid and specific to quantify these populations of bacteria. This study was performed to investigate the viability of adapting the indirect ELISA technique to quantify individually the populations of these three species of diazotroph within the root and shoot tissues of sugarcane. The results showed that species-specific polyclonal antibodies could be obtained by purifying sera in protein-A columns which removed non-specific immuno-globulins. It was possible to quantify the three bacterial species in the Brazilian sugarcane variety SP 70-1143 in numbers above 105 cells per g fresh weight in roots, rhizomes and leaves. The numbers of the different bacterial species evaluated using the ELISA technique were found to be higher than when the same populations were evaluated using the MPN technique, reaching 1400 times greater for G. diazotrophicus and 225 times greater for Herbaspirillum spp. These results constitute the first quantification of Herbaspirillum using immunological techniques.

A Leaf Spot and Blight of Greenhouse Tomato Seedlings Incited by a Herbaspirillum sp.

A leaf spot and blighting were observed on leaves of tomato transplants from a producer in Florida in 2001 and 2002. A nonfluorescent bacterium was isolated consistently from affected tissue. The typical bacterium was a gram negative, strictly aerobic, slightly curved rod with one or two flagella. Sequence analysis of the 16S rRNA indicated that two representative strains, F1 and SE1, had greater than 99% nucleotide sequence identity with Herbaspirillum huttiense and H. rubrisubalbicans. The cellular fatty acid composition of the total of 16 tomato strains was very similar to H. huttiense and H. rubrisubalbicans. Based on carbon utilization, six of nine strains tested with the Biolog system were identified as Herbaspirillum spp. The tomato strains were oxidase positive and grew at 40°C, but were negative for levan production, pectate hydrolysis, and arginine dihydrolase activity. Based upon this polyphasic analysis, we concluded that the strains were most closely related to H. huttiense, although placement in this species would require further analyses. However, the tomato strains and H. rubrisubalbicans, but not H. huttiense, caused confluent necrosis when infiltrated at high concentrations into tomato leaves and were able to produce leaf spot symptoms on inoculated tomato seedlings in the greenhouse. Using pulsed-field gel electrophoresis, we determined that there was considerable variability between the strains collected in 2001 and 2002.

Colonization ability of Herbaspirillum spp. B501gfp1 in sugarcane, a non-host plant in the presence of indigenous diazotrophic endophytes

Inoculating sugarcane with a mixture of diazotrophic endophytic bacteria has shown that they can provide substantial amount of biologically fixed nitrogen to the plant. The genera of diazotrophic endophytes previously isolated from sugarcane have been reported associating with other nonleguminous plants showing a broad host range. This study examined the colonization ability of a wild rice isolate, Herbaspirillum spp., in sugarcane plants in the presence of indigenous endophytes using two inoculum concentrations (102 and 108 bacterial cells ml-1). Internal tissue colonization was observed in plants inoculated with both the 102 and 108 B501gfp1 bacterial cells ml-1 inoculum concentrations. However, extensive colonization and higher bacterial numbers were determined only in the basal stem tissues of plants inoculated with the 108 bacterial cells ml-1.

Relação entre distribuição de nitrogênio e colonização por bactérias diazotróficas em cana-de-açúcar

O objetivo deste trabalho foi avaliar a relação entre a distribuição de nitrogênio na planta e a colonização por Gluconacetobacter diazotrophicus e Herbaspirillum spp. Foi implantado um experimento em parcelas subdivididas, tendo como tratamentos quatro genótipos: SP70-1143, SP79-2312 (híbridos), Krakatau (Saccharum spontaneum L.) e Chunnee (Saccharum barberi Jesw.); e quatro épocas de coleta: 90, 180, 360 e 540 dias após o plantio, com quatro repetições. Os parâmetros estudados foram: porcentual de nitrogênio, nitrato, N amino livre e o número mais provável de população das bactérias Gluconacetobacter diazotrophicus e Herbaspirillum spp. Os valores mais elevados de nitrogênio foram observados aos 90 dias após o plantio, nas folhas e colmo apical; as maiores concentrações N amino ocorreram nas raízes, colmo basal e folhas da variedade SP79-2312 e no colmo apical do genótipo Chunnee. O maior acúmulo de nitrato foi observado nos colmos basais e intermediários, sobretudo na variedade SP79-2312. O N protéico mostrou ser o maior componente do nitrogênio porcentual, com a mesma tendência nas diferentes partes da planta. O maior valor na população de G. diazotrophicus ocorreu nas raízes de SP70-1143, SP79-2312 e Krakatau, e nos quatro genótipos de Herbaspirillum spp.

Lower Extremity Cellulitis and Bacteremia With Herbaspirillum seropedicae Associated With Aquatic Exposure in a Patient With Cirrhosis

We report the first documented case of pathogenic human infection caused by Herbaspirillum spp, a class of gram-negative organisms related to Burkholderia spp, presenting as bacteremia and cellulitis in a patient with aquatic exposure. Herbaspirillum spp may be an under recognized human pathogen associated with freshwater exposure. Routine biochemical testing may be insufficient to rapidly distinguish the organism from more virulent aquaphilic organisms causing cellulitis. However, Herbaspirillum spp cellulitis responds well to common antimicrobial agents.

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

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

Herbaspirillum frisingense sp. nov., a new nitrogen-fixing bacterial species that occurs in C4-fibre plants.

The enrichment of nitrogen-fixing bacteria from the C4-fibre plants, Spartina pectinata, Miscanthus sinensis, Miscanthus sacchariflorus and Pennisetum purpureum, with nitrogen-free semi-solid media led to the isolation of Herbaspirillum-like strains among other diazotrophic bacteria. On the basis of physiological properties, phylogenetic analysis comparing 16S rDNA sequences and DNA-DNA hybridization experiments of chromosomal DNA the new isolates could be grouped together in a new species with the proposed name Herbaspirillum frisingense sp. nov. Morphological characteristics, such as cell size and shape, colony appearance, motility and flagellation are largely identical to the known species Herbaspirillum rubrisubalbicans and Herbaspirillum seropedicae. On the basis of utilization of adipate (-), N-acetyl-D-glucosamine (+), meso-erythritol (-), L-rhamnose (-) and meso-inositol (-) Herbaspirillum frisingense sp. nov. can be distinguished from other known Herbaspirillum spp. Nitrogen-fixing capability was examined by PCR amplification of the nifD gene and an acetylene reduction assay, and was found with all isolates tested. 16S rDNA sequence similarity to the other Herbaspirillum spp. is 98.5-99.1%. In genomic DNA-DNA hybridization experiments Herbaspirillum frisingense sp. nov. forms a homogeneous group with 70-100+/-10% similarity, clearly distinct from Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans with 1-34% similarity. 16S rRNA-targeted oligonucleotide probes, specific for the whole genus Herbaspirillum and for three Herbaspirillum species were designed and are suitable for fluorescence in situ hybridization. The DNA G+C content of Herbaspirillum frisingense sp. nov. is 63+/-2 mol%, in agreement with the values of 61-65% for the genus. PCR fingerprinting exhibits a consistent pattern for groups of strains isolated from the same plant, suggesting a low genomic diversity among bacteria inhabiting C4-gramineous plant tissues. Low genetic DNA diversity seems to be common between probable endophytic bacterial isolates of the same taxon. The type strain of Herbaspirillum frisingense sp. nov. is GSF30T (= DSM 13128T).

Recent advances in BNF with non-legume plants

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