Unraveling Azospirillum's colonization ability through microbiological and molecular evidence.

It is known that members of the bacterial genus Azospirillum can promote the growth of a great variety of plants, an ability harnessed by the industry to create bioproducts aimed to enhance the yield of economically relevant crops. Its versatile metabolism allows this bacterium to adapt to numerous environments, from optimal to extreme or highly polluted. The fact of having been isolated from soil and rhizosphere samples collected worldwide and many other habitats proves its remarkable ubiquity. Azospirillum rhizospheric and endophytic lifestyles are governed by several mechanisms, leading to efficient niche colonization. These mechanisms include cell aggregation and biofilm formation, motility, chemotaxis, phytohormone and other signaling molecules production, and cell-to-cell communication, in turn, involved in regulating Azospirillum interactions with the surrounding microbial community. Despite being infrequently mentioned in metagenomics studies after its introduction as an inoculant, an increasing number of studies detected Azospirillum through molecular tools (mostly 16S rRNA sequencing) as part of diverse, even unexpected, microbiomes. This review focuses on Azospirillum traceability and the performance of the available methods, both classical and molecular. An overview of Azospirillum occurrence in diverse microbiomes and the less-known features explaining its notorious ability to colonize niches and prevail in multiple environments is provided.

Azospirillum Aestuarii sp. nov., a Novel Nitrogen-Fixting and Aerobic Denitrifying Bacteria Isolated from an Estuary of a Freshwater River.

A novel Gram-staining negative, aerobic, rod-shaped bacterium, designated strain YIM DDC1T, was isolated from an estuary sediment sample of Dongda River flowing into Dianchi lake in Yunnan, southwest China. The strain displayed growth at 10-40 °C (optimum of 28 °C), pH 5.0-9.0 (optimum of 7.0-8.0) and in presence of 0-3% (w/v) NaCl (optimum of 0-1%). Strain YIM DDC1T comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and two unidentified aminolipids as the predominant polar lipids; the ubiquinone 10 as the major respiratory quinone; and summed feature 8 (C18:1ω6c and/or C18:1ω7c), summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C18:1 2-OH as the major cellular fatty acids. Analysis of 16S rRNA showed that YIM DDC1T represents a member of the genus Azospirillum, and was closely related to A. brasilense ATCC 29145 T (98.9%), A. baldaniorum Sp245T (98.2%), A. argentinense Az39T (98.2%) and A. formosense CC-Nfb-7 T (98.2%). The draft genome size was 7.15 Mbp with a 68.4% G + C content. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain YIM DDC1T and the aforementioned closely related strains exhibited similarity in the range of 93.8-93.5% and 53.7-52.7%, respectively. nif gene cluster (nifHDK) and denitrification genes ((napA, nirS, nirK, norBC and nosZ) detected in the genome indicated its potential nitrogen fixation and full-fledged denitrifying function. Based on combined genotypic and phenotypic data, strain YIM DDC1T represents a novel species of the genus Azospirillum, for which the name Azospirillum aestuarii sp. nov. is proposed. The type strain is YIM DDC1T (= KCTC 42887 T = CGMCC 1.17325 T).

Molecular Mechanisms Determining the Role of Bacteria from the Genus Azospirillum in Plant Adaptation to Damaging Environmental Factors.

Agricultural plants are continuously exposed to environmental stressors, which can lead to a significant reduction in yield and even the death of plants. One of the ways to mitigate stress impacts is the inoculation of plant growth-promoting rhizobacteria (PGPR), including bacteria from the genus Azospirillum, into the rhizosphere of plants. Different representatives of this genus have different sensitivities or resistances to osmotic stress, pesticides, heavy metals, hydrocarbons, and perchlorate and also have the ability to mitigate the consequences of such stresses for plants. Bacteria from the genus Azospirillum contribute to the bioremediation of polluted soils and induce systemic resistance and have a positive effect on plants under stress by synthesizing siderophores and polysaccharides and modulating the levels of phytohormones, osmolytes, and volatile organic compounds in plants, as well as altering the efficiency of photosynthesis and the antioxidant defense system. In this review, we focus on molecular genetic features that provide bacterial resistance to various stress factors as well as on Azospirillum-related pathways for increasing plant resistance to unfavorable anthropogenic and natural factors.

Inoculation with Azospirillum argentinense Az19 improves the yield of maize subjected to water deficit at key stages of plant development.

Water deficit constitutes a severe limitation to agricultural productivity. In the context of sustainable crop production, the potential of microbial biotechnology to increase plant drought tolerance and improve crop yields under adverse conditions is gaining relevance. This work aimed to compare the performance of Azospirillumargentinense strain Az19 to that of strain Az39, the most widely used for commercial inoculants, when inoculated in maize plants exposed to water deficit. For this purpose, greenhouse and field assays were conducted. In the greenhouse experiment, strain Az19 prevented the adverse effect of water deficit at V2 stage on maize growth. Moreover, the percentage of fertile plants and the ear weight decreased significantly under water deficits imposed at V2 and flowering in Az39-inoculated plants but not in Az19-inoculated plants. In the first field trial with the commercial maize hybrid DOW DS 515 PW, Az19-inoculated plants were those which better tolerated the water deficit imposed. In the second field trial, two maize genotypes with differential drought sensitivity (LP 29×LP 2542, sensitive; LP 882 (923)×LP 4703, tolerant) were tested. Higher tolerance to water deficit was detected in plants inoculated with A. argentinense Az19, with a noticeable effect on grain yield components in the sensitive genotype. Based on these results, we propose the use of A. argentinense Az19 for the formulation of more targeted Azospirillum-based inoculants, suitable for agroecological areas subjected to seasonal water deficits.

ß-Lactam Resistance in Azospirillum baldaniorum Sp245 Is Mediated by Lytic Transglycosylase and ß-Lactamase and Regulated by a Cascade of RpoE7→RpoH3 Sigma Factors.

Bacterial resistance to ß-lactam antibiotics is often mediated by ß-lactamases and lytic transglycosylases. Azospirillum baldaniorum Sp245 is a plant-growth-promoting rhizobacterium that shows high levels of resistance to ampicillin. Investigating the molecular basis of ampicillin resistance and its regulation in A. baldaniorum Sp245, we found that a gene encoding lytic transglycosylase (Ltg1) is organized divergently from a gene encoding an extracytoplasmic function (ECF) σ factor (RpoE7) in its genome. Inactivation of rpoE7 in A. baldaniorum Sp245 led to increased ability to form cell-cell aggregates and produce exopolysaccharides and biofilm, suggesting that rpoE7 might contribute to antibiotic resistance. Inactivation of ltg1 in A. baldaniorum Sp245, however, adversely affected its growth, indicating a requirement of Ltg1 for optimal growth. The expression of rpoE7, as well that of as ltg1, was positively regulated by RpoE7, and overexpression of RpoE7 conferred ampicillin sensitivity to both the rpoE7::km mutant and its parent. In addition, RpoE7 negatively regulated the expression of a gene encoding a ß-lactamase (bla1). Out of the 5 paralogs of RpoH encoded in the genome of A. baldaniorum Sp245, RpoH3 played major roles in conferring ampicillin sensitivity and in the downregulation of bla1. The expression of rpoH3 was positively regulated by RpoE7. Collectively, these observations reveal a novel regulatory cascade of RpoE7-RpoH3 σ factors that negatively regulates ampicillin resistance in A. baldaniorum Sp245 by controlling the expression of a ß-lactamase and a lytic transglycosylase. In the absence of a cognate anti-sigma factor, addressing how the activity of RpoE7 is regulated by ß-lactams will unravel new mechanisms of regulation of ß-lactam resistance in bacteria. IMPORTANCE Antimicrobial resistance is a global health problem that requires a better understanding of the mechanisms that bacteria use to resist antibiotics. Bacteria inhabiting the plant rhizosphere are a potential source of antibiotic resistance, but their mechanisms controlling antibiotic resistance are poorly understood. A. baldaniorum Sp245 is a rhizobacterium that is known for its characteristic resistance to ampicillin. Here, we show that an AmpC-type ß-lactamase and a lytic transglycosylase mediate resistance to ampicillin in A. baldaniorum Sp245. While the gene encoding lytic transglycosylase is positively regulated by an ECF σ-factor (RpoE7), a cascade of RpoE7 and RpoH3 σ factors negatively regulates the expression of ß-lactamase. This is the first evidence showing involvement of a regulatory cascade of σ factors in the regulation of ampicillin resistance in a rhizobacterium.

Co-inoculation with tropical strains of Azospirillum and Bacillus is more efficient than single inoculation for improving plant growth and nutrient uptake in maize.

Usage of Bacillus and Azospirillum as new eco-friendly microbial consortium inoculants is a promising strategy to increase plant growth and crop yield by improving nutrient availability in agricultural sustainable systems. In this study, we designed a multispecies inoculum containing B. thuringiensis (strain B116), B. subtillis (strain B2084) and Azospirillum sp. (strains A1626 and A2142) to investigate their individual or co-inoculated ability to solubilize and mineralize phosphate, produce indole acetic acid (IAA) and their effect on maize growth promotion in hydroponics and in a non-sterile soil. All strains showed significant IAA production, P mineralization (sodium phytate) and Ca-P, Fe-P (tricalcium phosphate and iron phosphate, respectively) solubilization. In hydroponics, co-inoculation with A1626 x A2142, B2084 x A2142, B2084 x A1626 resulted in higher root total length, total surface area, and surface area of roots with diameter between 0 and 1 mm than other treatments with single inoculant, except B2084. In a greenhouse experiment, maize inoculated with the two Azospirillum strains exhibited enhanced shoot dry weight, shoot P and K content, root dry weight, root N and K content and acid and alkaline phosphatase activities than the other treatments. There was a significant correlation between soil P and P shoot, alkaline phosphatase and P shoot and between acid phosphatase and root dry weight. It may be concluded that co-inoculations are most effective than single inoculants strains, mainly between two selected Azospirillum strains. Thus, they could have synergistic interactions during maize growth, and be useful in the formulation of new inoculants to improve the tropical cropping systems sustainability.

Engineering D-glucose utilization in Azospirillum brasilense Sp7 promotes rice root colonization.

Bacteria of the genus Azospirillum include several plant associated bacteria which often promote the growth of their host plants. Although the host range of Azospirillum brasilense Sp7 is much wider than its close relative Azospirillum lipoferum 4B, it lacks the ability to efficiently utilize D-glucose for its growth. By comparing the genomes of both the species, the genes of A. lipoferum 4B responsible for conferring D-glucose utilization ability in A. brasilese Sp7 were identified by cloning individual or a combination of genes in a broad host range expression vector, mobilizing them in A. brasilense Sp7 and examining the ability of exconjugants to use D-glucose as sole carbon source for growth. These genes also included the homologs of genes involved in N-acetyl glucosamine utilization in Pseudomonas aeruginosa PAO1. A transcriptional fusion of the 5 genes encoding glucose-6-phosphate dehydrogenase and 4 components of glucose phosphotransferase system were able to improve D-glucose utilization ability in A. brasilense Sp7. The A. brasilense Sp7 strain engineered with D-glucose utilization ability showed significantly improved root colonization of rice seedling. The improvement in the ability of A. brasilense Sp7 to colonize rice roots is expected to bring benefits to rice by promoting its growth. KEY POINTS: • Genes required for glucose utilization in Azospirillum lipoferum were identified. • A gene cassette encoding glucose utilization was constructed. • Transfer of gene cassette in A. brasilense improves glucose utilization and rice root colonization..

Azospirillum Endophyticum sp. nov., an Endophyte of Paris Polyphylla Smith var. Yunnanensis.

A Gram-negative, facultative anaerobic bacterial strain, designated YIM B02556T, was isolated from the root of Paris polyphylla Smith var. yunnanensis collected from Yunnan Province, southwest China. By using a polyphasic approach, its taxonomic position was investigated. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YIM B02556T belonged to the genus Azospirillum and the 16S rRNA gene sequence similarity values of strain YIM B02556T to the type strains of members of this genus ranged from 94.9 to 98.3%. Overall genome relatedness index (OGRI) analysis estimated based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between YIM B02556T and other Azospirillum species type strains were <90.8% and <37.8%, lower than the limit of species circumscription. Cells of the strain were characterized as oxidase- and catalase-positive, with motility provided by flagella. The growth conditions of the strain were found to occur at 20-40 °C (optimum, 35 °C), and pH 6.0-9.5 (optimum, pH 7.5). Strain YIM B02556T can tolerate 2% NaCl concentration. Strain YIM B02556T contained Q-10 as the major ubiquinone. The major fatty acids were C18:1 ω7c and summed feature three (C16:1 ω7c and/or C16:1 ω6c). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. Based on polyphasic analysis, strain YIM B02556T could be differentiated genotypically and phenotypically from recognized species of the genus Azospirillum. Therefore, the isolate represents a novel species, for which the name Azospirillum endophyticum is proposed. The type strain is YIM B02556T (=JCM 34631T=CGMCC 1.18654T).

Behavior and interactions of the plant growth-promoting bacteria Azospirillum oryzae NBT506 and Bacillus velezensis UTB96 in a co-culture system.

The objective of the present study was to evaluate possible interactions between two potential plant growth-promoting bacteria (PGPB): Azospirillum oryzae strain NBT506 and Bacillus velezensis strain UTB96. To do this, the growth kinetic, biofilm formation, motility, surfactin production, indole-3-acetic acid (IAA) production, phosphate solubilization and enzyme activities of the strains were measured in monoculture and co-culture. The maximum biomass production for the strains in monoculture and co-culture was about 1011 CFU/ml, confirming that these two strains have the potential to grow in co-culture without reduction of biomass efficiency. The co-culture system showed more stable biofilm formation until the end of day 3. Azospirillum showed the maximum IAA production (41.5 mg/l) in a monoculture compared to other treatments. Surfactin promoted both swimming and swarming motility in all treatments. The Bacillus strain in the monoculture and co-culture showed high phosphate solubilizing capability, which increased continuously in the co-culture system after 6 days. The strains showed protease, amylase and cellulase activities in both monoculture and co-culture forms. Chitinase and lipase activities were observed in both the monoculture of the Bacillus strain and the co-culture. Overall, our findings highlight the promotion of biological and beneficial effects of these bacteria when growing together in co-culture.

The drop plate method as an alternative for Azospirillum spp. viable cell enumeration within the consensus protocol of the REDCAI network.

Quality evaluation of commercial inoculants is essential to warrant an adequate crop response to inoculation within a biosecurity framework. In this sense, this work is aimed at standardizing and validating the drop plate method for the enumeration of Azospirillum viable cells as an alternative to the spread plate technique, which is currently proposed in the consensus protocol of the REDCAI network. Between 14 and 25 private and public laboratories participated in three independent trials. We obtained consistent and robust results that allowed to confirm that both techniques are equivalent, concluding that the drop plate method is an alternative enumeration technique that is adequate to be included in the abovementioned consensus protocol.

Azospirillum tabaci sp. nov., a bacterium isolated from rhizosphere soil of Nicotiana tabacum L.

Strain W712T was isolated from rhizosphere soil of Nicotiana tabacum L. collected from Kunming, south-west China. Cells were Gram-staining negative, aerobic, motile and rod shaped. The isolate grew at 20-45 °C (optimum 30 °C), pH 6.0-8.0 (optimum pH 7.0) and in the presence of up to 3.0% (w/v) NaCl (optimum 1%, w/v). Ubiquinone-10 was the only respiratory quinone type. Polar lipids contained diphosphatidylglycerol, phosphatidylmehtylethanolamine, phosphatidylglycerol, phosphatidylcholine and an unidentified aminolipid. The major fatty acids were detected as summed feature 8 (C18:1 ω7c or C18:1 ω6c), summed feature 3 (C16:1 ω7c or C16:1 ω6c) and C18:1 2OH. The genomic DNA G + C content was 68.7%. The ANI values were 94.3%, 93.3% and 93.6% between Azospirillum baldaniorum Sp245T, Azospirillum brasilense ATCC 49958T, Azospirillum formosense CC-Nfb-7T and strain W712T, respectively, which were lower than the prokaryotic species delineation threshold of 95.0-96.0%. The digital DNA-DNA hybridization values between A. baldaniorum Sp245T, A. brasilense ATCC 49958T, A. formosense CC-Nfb-7T and strain W712T indicated that the candidate represents a novel genomic species. According to the phenotypic and genotypic characteristics, we propose that strain W712T warrants the assignment to a novel species, for which the name Azospirillum tabaci sp. nov. (type strain W712T = CGMCC 1.18567T = KCTC 82186T) is proposed.

What Do We Know About the Publications Related with Azospirillum? A Metadata Analysis.

Azospirillum is one of the most successful plant growth-promoting bacteria (PGPB) genera and it is considered a study model for plant-bacteria interactions. Because of that, a wide broad of topics has been boarded and discussed in a significant number of publications in the last four decades. Using the Scopus® database, we conducted a bibliographic search in order to analyze the number and type of publications, the authors responsible of these contributions, and the origin of the researchers, as well as the keywords and journals selected by the authors, among other related characteristics, with the aim to understand some less addressed details about the work done with Azospirillum worldwide since its discovery in 1925. Despite that the largest numbers of publications about this bacterium were obtained between the 1970 and 1980s, there is still a linear increase tendency in the number of published works. Understanding the mechanisms involved in the ability of these bacteria to promote growth in a wide broad of plant species under both laboratory and field conditions has been a preferential target for these published articles. This tendency could be considered a cause or consequence of the current increase in the number of commercial products formulated with Azospirillum around the world and a catalyzer for the increase of published articles along time.

Bacterial Diversity in a Sri Lankan Geothermal Spring Assessed by Culture-Dependent and Culture-Independent Approaches.

Hot springs harbour diverse and interesting groups of microorganisms adapted to extreme conditions. However, due to limitations in the culture-dependent approach, most of such thermophiles remain uncultured and unexplored. Hence, this study was conducted to gain a comprehensive understanding of the bacterial diversity of Mahapelessa hot spring, Sri Lanka using both culture-dependent and culture-independent approaches. The in situ temperature of the water sample was 44.5 °C and the pH was 8.14. 16S rRNA Sanger sequencing of DNA extracted from the 18 bacterial isolates revealed the presence of eight genera belonging to two phyla: Proteobacteria (84%) and Firmicutes (16%) and the most abundant genus being Klebsiella. A total of 23 bacterial phyla representing 80 classes, 43 orders, 123 families, 205 genera and 83 species were detected by 16S rRNA V3-V4 region by amplicon metagenome sequencing of DNA extracted from water samples, where the most abundant phylum was the Proteobacteria (57.39%), followed by Firmicutes (23.7%) and Chloroflexi (4.14%). The three phyla Actinobacteria, Planctomycetes and Bacteroidetes were also detected less than 3% in abundance while 4.48% of bacteria could not be fit into any known phylum. The most abundant genera were Burkholderia (14.87%), Desulfotomaculum (7.23%) and Stenotrophomonas (6.1%). Four strictly anaerobic bacteria, Anaerosolibacter carboniphilus (0.71%), Bellilinea caldifistulae (0.04%), Salimesophilobacter vulgaris (0.1%), Anaerobacterium chartisolvens (0.12%); two potential plant growth-promoting bacteria, Azospirillum halopraeferens (0.04%) and Bradyrhizobium liaoningense (0.16%) and one potential alkali tolerant and sulphate-reducing bacterium, Desulfovibrio alkalitolerans (0.45%) were recorded. Pigmentiphaga sp. was isolated from Mahapelessa hot spring and to the best of our knowledge, this is the first record of this genus from a hot spring. This study gives insight into the vast bacterial diversity present in the Mahapelessa hot spring from the culture-independent approach which could not be identified using standard culturing techniques.

Fourier Transform Infrared (FTIR) Spectroscopic Analyses of Microbiological Samples and Biogenic Selenium Nanoparticles of Microbial Origin: Sample Preparation Effects.

To demonstrate the importance of sample preparation used in Fourier transform infrared (FTIR) spectroscopy of microbiological materials, bacterial biomass samples with and without grinding and after different drying periods (1.5-23 h at 45 °C), as well as biogenic selenium nanoparticles (SeNPs; without washing and after one to three washing steps) were comparatively studied by transmission FTIR spectroscopy. For preparing bacterial biomass samples, Azospirillum brasilense Sp7 and A. baldaniorum Sp245 (earlier known as A. brasilense Sp245) were used. The SeNPs were obtained using A. brasilense Sp7 incubated with selenite. Grinding of the biomass samples was shown to result in slight downshifting of the bands related to cellular poly-3-hydroxybutyrate (PHB) present in the samples in small amounts (under ~10%), reflecting its partial crystallisation. Drying for 23 h was shown to give more reproducible FTIR spectra of bacterial samples. SeNPs were shown to contain capping layers of proteins, polysaccharides and lipids. The as-prepared SeNPs contained significant amounts of carboxylated components in their bioorganic capping, which appeared to be weakly bound and were largely removed after washing. Spectroscopic characteristics and changes induced by various sample preparation steps are discussed with regard to optimising sample treatment procedures for FTIR spectroscopic analyses of microbiological specimens.

Towards sustainable yield improvement: field inoculation of soybean with Bradyrhizobium and co-inoculation with Azospirillum in Mozambique.

The effects of sole inoculation of soybean (Glycine max L. Merrill) with Bradyrhizobium and co-inoculation with Bradyrhizobium and Azospirillum on nodulation, plant growth and yields were investigated in the 2013/2014 and 2014/2015 cropping seasons under field conditions in Mozambique. The treatments included (1) Control (non-inoculated control, with symbiosis depending on indigenous rhizobia), (2) Urea (non-inoculated, receiving 200 kg ha-1 of N), (3) Sole inoculation with B. diazoefficiens strain USDA 110, and (4) Co-inoculation with B. diazoefficiens strain USDA 110 and A. brasilense strains Ab-V5 and Ab-V6, evaluated in a randomized complete block design with five replications. Nodule number and dry weight, shoot dry weight, biological and grain yields, grain dry weight, and harvest index were evaluated. In general, both sole inoculation and co-inoculation enhanced nodulation in relation to control. Sole inoculation increased grain yield by 22% (356 kg ha-1), the same enhancement magnitude attained under mineral N treatment, suggesting that Bradyrhizobium inoculation provides ecological and economic sustainability to the soybean crop in Mozambique or other countries with similar agro-climatic conditions. Co-inoculation did not increase grain yields in relation to neither the control nor sole inoculation, indicating that further research with adapted and high yielding soybean varieties along with effective rhizobial strains is required in Mozambique to attune the beneficial Azospirillum-plant cultivar-rhizobia interactions that have been reported in other countries for several legumes, including soybean.

Azospirillum thermophilum sp. nov., isolated from a hot spring.

A novel Gram-staining negative, aerobic, motile by flagellum, rod-shaped bacterium, designated CFH 70021T was isolated from a hot spring soil sample collected from Tengchong, Yunnan province, PR China. Growth of CFH 70021T occurred at 15-50 °C (optimum 50 °C), pH 5.0-7.0 (optimum pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum 0 %, w/v). The genome of CFH 70021T consisted of four complete circular chromosomes and five plasmids, the genomic DNA G+C content was 69.3 mol%. Comparison of the 16S rRNA gene sequences indicated that CFH 70021T represented a member of the genus Azospirillum and showed close relationship with the type strains of Azospirillum agricola CC-HIH038T (97.8 %), Azospirillum rugosum IMMIB AFH-6T (97.6 %), Azospirillum doebereinerae GSF71T (97.6 %), Azospirillum thiophilum DSM 21654T (97.4 %) and Azospirillum picis IMMIB TAR-3T (97.2 %). The polar lipids of CFH 70021T contained diphosphatidylglycerol, phosphatidylmehtylethanolamine, phosphatidylglycerol, phosphatidylcholine, two aminolipids and an unidentified phospholipid. The predominant cellular fatty acids (>10 %) included C19:0cyclo ω8c (11.4 %), C16 : 0 (27.6 %) and summed feature 8 (C18:1ω7c/C18:1ω6c, 40.9 %). The major isoprenoid quinone was Q-10. On the basis of the low ANIb result (<78 %) and different phenotypic and chemotaxonomic characters, we conclude that strain CFH 70021T represents a novel member of the genus Azospirillum, for which the name Azospirillum thermophilum sp. nov. is proposed. The type strain is CFH 70021T (=KCTC 62259T= CCTCC AB2018121T).

Genome-based reclassification of Azospirillum brasilense Sp245 as the type strain of Azospirillum baldaniorum sp. nov.

Azospirillum sp. strain Sp245T, originally identified as belonging to Azospirillum brasilense, is recognized as a plant-growth-promoting rhizobacterium due to its ability to fix atmospheric nitrogen and to produce plant-beneficial compounds. Azospirillum sp. Sp245T and other related strains were isolated from the root surfaces of different plants in Brazil. Cells are Gram-negative, curved or slightly curved rods, and motile with polar and lateral flagella. Their growth temperature varies between 20 to 38 °C and their carbon source utilization is similar to other Azospirillum species. A preliminary 16S rRNA sequence analysis showed that the new species is closely related to A. brasilense Sp7T and A. formosense CC-Nfb-7T. Housekeeping genes revealed that Azospirillum sp. Sp245T, BR 12001 and Vi22 form a separate cluster from strain A. formosense CC-Nfb-7T, and a group of strains closely related to A. brasilense Sp7T. Overall genome relatedness index (OGRI) analyses estimated based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between Azospirillum sp. Sp245T and its close relatives to other Azospirillum species type strains, such as A. brasilense Sp7T and A. formosense CC-Nfb-7T , revealed values lower than the limit of species circumscription. Moreover, core-proteome phylogeny including 1079 common shared proteins showed the independent clusterization of A. brasilense Sp7T, A. formosense CC-Nfb-7T and Azospirillum sp. Sp245T, a finding that was corroborated by the genome clustering of OGRI values and housekeeping phylogenies. The DNA G+C content of the cluster of Sp245T was 68.4-68.6 %. Based on the phylogenetic, genomic, phenotypical and physiological analysis, we propose that strain Sp245T together with the strains Vi22 and BR12001 represent a novel species of the genus Azospirillum, for which the name Azospirillum baldaniorum sp. nov. is proposed. The type strain is Sp245T (=BR 11005T=IBPPM 219T) (GCF_007827915.1, GCF_000237365.1, and GCF_003119195.2).

In vitro characterization of root extracellular trap and exudates of three Sahelian woody plant species.

MAIN CONCLUSION: Arabinogalactan protein content in both root extracellular trap and root exudates varies in three Sahelian woody plant species that are differentially tolerant to drought. At the root tip, mature root cap cells, mainly border cells (BCs)/border-like cells (BLCs) and their associated mucilage, form a web-like structure known as the "Root Extracellular Trap" (RET). Although the RET along with the entire suite of root exudates are known to influence rhizosphere function, their features in woody species is poorly documented. Here, RET and root exudates were analyzed from three Sahelian woody species with contrasted sensitivity to drought stress (Balanites aegyptiaca, Acacia raddiana and Tamarindus indica) and that have been selected for reforestation along the African Great Green Wall in northern Senegal. Optical and transmission electron microscopy show that Balanites aegyptiaca, the most drought-tolerant species, produces only BC, whereas Acacia raddiana and Tamarindus indica release both BCs and BLCs. Biochemical analyses reveal that RET and root exudates of Balanites aegyptiaca and Acacia raddiana contain significantly more abundant arabinogalactan proteins (AGPs) compared to Tamarindus indica, the most drought-sensitive species. Root exudates of the three woody species also differentially impact the plant soil beneficial bacteria Azospirillum brasilense growth. These results highlight the importance of root secretions for woody species survival under dry conditions.

Potentially Mobile Denitrification Genes Identified in Azospirillum sp. Strain TSH58.

Denitrification ability is sporadically distributed among diverse bacteria, archaea, and fungi. In addition, disagreement has been found between denitrification gene phylogenies and the 16S rRNA gene phylogeny. These facts have suggested potential occurrences of horizontal gene transfer (HGT) for the denitrification genes. However, evidence of HGT has not been clearly presented thus far. In this study, we identified the sequences and the localization of the nitrite reductase genes in the genomes of 41 denitrifying Azospirillum sp. strains and searched for mobile genetic elements that contain denitrification genes. All Azospirillum sp. strains examined in this study possessed multiple replicons (4 to 11 replicons), with their sizes ranging from 7 to 1,031 kbp. Among those, the nitrite reductase gene nirK was located on large replicons (549 to 941 kbp). Genome sequencing showed that Azospirillum strains that had similar nirK sequences also shared similar nir-nor gene arrangements, especially between the TSH58, Sp7T, and Sp245 strains. In addition to the high similarity between nir-nor gene clusters among the three Azospirillum strains, a composite transposon structure was identified in the genome of strain TSH58, which contains the nir-nor gene cluster and the novel IS6 family insertion sequences (ISAz581 and ISAz582). The nirK gene within the composite transposon system was actively transcribed under denitrification-inducing conditions. Although not experimentally verified in this study, the composite transposon system containing the nir-nor gene cluster could be transferred to other cells if it is moved to a prophage region and the phage becomes activated and released outside the cells. Taken together, strain TSH58 most likely acquired its denitrification ability by HGT from closely related Azospirillum sp. denitrifiers.IMPORTANCE The evolutionary history of denitrification is complex. While the occurrence of horizontal gene transfer has been suggested for denitrification genes, most studies report circumstantial evidences, such as disagreement between denitrification gene phylogenies and the 16S rRNA gene phylogeny. Based on the comparative genome analyses of Azospirillum sp. denitrifiers, we identified denitrification genes, including nirK and norCBQD, located on a mobile genetic element in the genome of Azospirillum sp. strain TSH58. The nirK was actively transcribed under denitrification-inducing conditions. Since this gene was the sole nitrite reductase gene in strain TSH58, this strain most likely benefitted by acquiring denitrification genes via horizontal gene transfer. This finding will significantly advance our scientific knowledge regarding the ecology and evolution of denitrification.

Azospirillum palustre sp. nov., a methylotrophic nitrogen-fixing species isolated from raised bog.

Nitrogen-fixing bacterial strain, designated B2T, was isolated from methane-oxidation enrichment originating from a Sphagnum-dominated raised peatland in Tver region, Russia, and its phenotypic, chemotaxonomic and genomic characteristics were investigated. Cells of isolate were Gram-negative, aerobic, rod or spiral-shaped, with motility provided by a single polar flagellum in liquid media and peritrichous flagella on solid media. Strain was able to grow at 15-40 °C, pH 5.5-8.5 and tolerated NaCl to 2.0 % (w/v). Strain B2T gave positive amplification for dinitrogen reductase (nifH gene) and acetylene reduction activity was recorded up to 1250 nmol ethylene h-1 (mg protein)-1. Analysis of 16S rRNA showed that B2T represents a member of the genus Azospirillum and had the highest sequence similarity with A. humicireducens SgZ-5T (97.92 %). The predominant quinone system was ubiquinone Q-10 and the major fatty acids were C18 : 1ω7, C16 : 1ω7 and C16 : 0. The strain was facultative methylotrophic and used methanol and formate for the growth. Genome sequencing revealed a genome size of 8.0 Mbp and a G+C content of 67.8 mol%. The mxaFI genes encoding methanol dehydrogenase were absent, but a homologous xoxF gene was detected. The genes encoding enzymes involved in the biosynthesis of tetrahydromethanopterin (H4MPT) (formaldehyde oxidation) and NAD-linked formate dehydrogenase (fdsABG) were identified. Pairwise determined whole genome average nucleotide identity (gANI) values confirmed that strain B2T represents a novel species, for which we propose the name Azospirillum palustre sp. nov. with the type strain B2T (VKM B-3233T, КСТС 62613Т).