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Rahnella aquatilis, a nitrogen-fixing enteric bacterium associated with the rhizosphere of wheat and maize
Canadian Journal of Microbiology
Abstract
In a study of dominant diazotrophic bacteria present in the rhizosphere of wheat and maize, 28 strains of Enterobacteriaceae were isolated. They were all Voges-Proskauer positive, motile at 28 °C but not at 37 °C, and they produced a Tween-80 esterase and did not exhibit decarboxylase activity. This fits well with the description of Rahnella aquatilis. The ability of these strains to reduce acetylene in pure culture and in association with their host plant and the DNA hybridization with a nifHDK probe are described. This is the first time that R. aquatilis is reported as a rhizosphere-associated bacterium and also a nitrogen fixer. Key words: Rahnella aquatilis, rhizosphere, wheat, maize, nitrogen fixation.
... Rahnella aquatilis was first studied and described in 1976 by the Pasteur Institute. Rahnella belongs to the Yersiniaceae family and is a Gram-negative, rod-shaped, fast-moving microaerophilic bacterium that is common in various environments (Adeolu, 1998), i.e. in soil, plant phyllosphere, water and food (Berge et al., 1991;Rhodes et al., 1998). There are currently 6 species of bacteria belonging to the Rahnella genus, namely R. aquatilis, R. variigena, R. inusitata, R. bruchi, R. woolbedingensis and R. victoriana. ...
... Of them, isolates UT3, UT4 and UT9 showed high acetylene-reducing activity (79-91 nmol C 2 H 4 /flacon/24h). Berge et al. (1991) reported the R. aquatilis CF1, CF3, CF4, TR01, TR02, and TR03 rhizospheric strains exerted acetylene reductase activity measuring 60-306 nmol С 2 Н 4 /tube (Berge et al., 1991). It should be noted that those strains of rhizobacteria also showed growth on the Ashby's nitrogen-free nutrient medium, which means that their nitrogen-fixing properties have been confirmed once again. ...
... Of them, isolates UT3, UT4 and UT9 showed high acetylene-reducing activity (79-91 nmol C 2 H 4 /flacon/24h). Berge et al. (1991) reported the R. aquatilis CF1, CF3, CF4, TR01, TR02, and TR03 rhizospheric strains exerted acetylene reductase activity measuring 60-306 nmol С 2 Н 4 /tube (Berge et al., 1991). It should be noted that those strains of rhizobacteria also showed growth on the Ashby's nitrogen-free nutrient medium, which means that their nitrogen-fixing properties have been confirmed once again. ...
As the number of people on earth increases, so does the need for food. Providing the population with environmentally friendly agricultural food is one of the urgent problems of our time. Currently, the main direction of modern organic farming is the use of biofertilizers. Bacterial preparations are capable of influencing the physiological processes of plants in small quantities, leading to increase in plant productivity. The objective of this work was studying rhizobac-teria associated with wheat roots. For this purpose, more than 100 isolates of rhizobacteria from the rhizosphere and root surface of wheat plants grown in irrigated fields of Tashkent, Syrdarya, Andijan, Kashkadarya regions. Rhizobac-teria were grown on nutrient media of Döbereiner, Ashby, Pikovsky, and Zack, and 25 isolates of associative rhizobac-teria were selected based on the characteristics of absorption of molecular nitrogen, mobilization of phosphorus and potassium. They actively dissolved Сa 3 (PO 4) 2 and KAlSiO 4 for 3 days. They were found to produce organic acids. In organic farming, nitrogen-fixing, phosphorus-and potassium-mobilizing rhizobacteria are of great practical importance , while our experiments on obtaining biological products are considered as an environmentally friendly and cost-effective way to increase crop yields. From the surface of wheat roots grown in different zones of Uzbekistan, when screening for nitrogen fixation, we selected 3 isolates with acetylene reductase activity of 79-91 nmol C 2 H 4 /flacon/24h. We determined that bacteria completely mobilized phosphate, forming 100% acid when grown in a medium containing Ca 3 (PO 4) 2 for 5 days. The ability of the bacteria to mobilize potassium was studied on a nutrient KAlSiO 4-containing medium. The bacteria were observed to mobilize potassium, forming 90-100% acid within 15 days. Based on the study of the 16S rRNA gene of bacteria, we identified rhizobacteria UT3, UT4, and UT9 as Rah-nella aquatilis.
... Rahnella was a Gram-negative, rod-shaped, facultatively anaerobic bacterium belonging to the family Yersiniaceae (Adeolu et al., 2016), and was widely distributed in a variety of environments, including soil, phyllosphere, water, seeds, food, some clinical samples, and even in American mastodon remains (Berge et al., 1991;Rhodes et al., 1998). Rahnella was first proposed in 1979, and normally consisted of six species including R. aquatilis (which was described as the type species of Rahnella), R. variigena, R. inusitata, R. bruchi, R. woolbedingensis, and R. victoriana by multilocus sequence analysis (Brady et al., 2014). ...
... Rahnella spp. were also reported to possess the capacity for nitrogen fixation (Berge et al., 1991). For, insurance, R. aquatilis HX2 was reported as a PGPR and could promote the growth of corn for the ability to fix nitrogen (Guo et al., 2012). ...
Many Rahnella strains have been widely described as plant growth-promoting rhizobacteria with the potential to benefit plant growth and protect plants from pathogens. R. aceris ZF458 is a beneficial plant bacterium isolated from swamp soil with the potential for biocontrol. Strain ZF458 has shown broad-spectrum antagonistic activities against a variety of plant pathogens and exhibited a dramatic effect on controlling Agrobacterium tumefaciens in sunflowers. The R. aceris ZF458 genome sequence contained a 4,861,340-bp circular chromosome and two plasmids, with an average G + C content of 52.20%. Phylogenetic analysis demonstrated that R. aceris ZF458 was closely related to R. aceris SAP-19. Genome annotation and comparative genomics identified the conservation and specificity of large numbers of genes associated with nitrogen fixation, plant growth hormone production, organic acid biosynthesis and pyrroloquinoline quinone production that specific to benefiting plants in strain ZF458. In addition, numerous conserved genes associated with environmental adaption, including the bacterial secretion system, selenium metabolism, two-component system, flagella biosynthesis, chemotaxis, and acid resistance, were also identified in the ZF458 genome. Overall, this was the first study to systematically analyze the genes linked with plant growth promotion and environmental adaption in R. aceris. The aim of this study was to derive genomic information that would provide an in-depth insight of the mechanisms of plant growth-promoting rhizobacteria, and could be further exploited to improve the application of R. aceris ZF458 in the agriculture field.
... These microbes have been already reported as rhizopsheric bacteria associated with different plants except Erwinia aphidicola. In a report, R. aquatilis CF3 was isolated from the rhizosphere of wheat plant [37]. Similarly, R. aquatilis ISL19 from rhizosphere of soybean [38]. ...
... R. aquatilis was originally isolated from freshwater samples in France but has also been isolated from the soil, the rhizosphere, and the intestine of snails; occasionally, it has been isolated from human clinical specimens and food (Kämpfer, 2015;Asselin et al., 2019). Additionally, R. aquatilis has been reported as a nitrogen-fixing (Berge et al., 1991) bacterium and a biocontrol agent (Chen et al., 2009). R. aceris, originally isolated from Acer pictum sap in the Republic of Korea (Lee et al., 2020), has also been isolated from onion bulbs with symptoms of bacterial decay in Norway and the United States of America (Brady et al., 2022) and from bleeding cankers of black poplar (Populus nigra) in Iran, with pathogenicity demonstrated by the formation of water-soaked cankers on this host (Moradi-Amirabad and Khodakaramian, 2020). ...
Rah'nel.la. L. fem. dim. n. suff. ‐ella , diminutive ending; N.L. fem. dim. n. Rahnella , named after Otto Rahn, the German‐American microbiologist who proposed the name Enterobacteriaceae in 1937.
Pseudomonadota / Gammaproteobacteria / Enterobacteriales / Enterobacteriaceae / Rahnella
Rahnella species are ubiquitous in the environment, regularly isolated from water, soil, and woody tree hosts as well as occasionally from human clinical samples. Cells are Gram stain negative, facultatively anaerobic, and motile rods. Species are psychrotolerant, able to grow at 4°C, and favor growth temperatures of 25–30°C. Carbon sources such as d ‐maltose, N ‐acetyl‐ d ‐glucosamine, α‐ d ‐glucose, and d ‐mannose are readily assimilated. Species form a monophyletic clade when phylogenetic analysis is based on multilocus sequence analysis (MLSA) or whole‐genome sequences. The major fatty acids are C 16:0 and C 17:0 cyclo; the percentage of C 18:1 ω7 c , summed feature 2 (iso‐C 16:1 and/or C 14:0 3‐OH), and summed feature 3 (C 16:1 ω7 c and/or iso‐C 15:0 2‐OH) is variable, with some species exhibiting these fatty acids in larger amounts.
DNA G + C content (mol%) : 51.3–53.7 (genome analysis).
Type species : Rahnella aquatilis Izard et al. 1979, VL7.
... These eight species contributed to the existing diversity of Rahnella with isolations of Rahnella victoriana, Rahnella variigena and Rahnella inusitata from bleeding cankers of oak; R. victoriana, R. variigena and R. woolbedingensis from asymptomatic alder and walnut; Rahnella bruchi from the gut of the Agrilus biguttatus beetle; Rahnella aceris and Rahnella laticis from sap of Acer pictum and Rahnella contaminans as a contaminant from MRSA agar plates [5][6][7]. In addition to their isolation from the natural environment, Rahnella species have been linked to nitrogen-fixation [8], metal and radionuclide sequestration [9] and biological control [10]; and more recently as possible pathogens of oak [11], poplar [12] and onion [13]. ...
Bacteria isolated from onion bulbs suffering from bacterial decay in the United States and Norway were previously shown to belong to the genus Rahnella based on partial housekeeping gene sequences and/or fatty acid analysis. However, many strains could not be assigned to any existing Rahnella species. Additionally, strains isolated from creek water and oak as well as a strain with bioremediation properties were assigned to Rahnella based on partial housekeeping gene sequences. The taxonomic status of these 21 strains was investigated using multilocus sequence analysis, whole genome analyses, phenotypic assays and fatty acid analysis. Phylogenetic and phylogenomic analyses separated the strains into five clusters, one of which corresponded to Rahnella aceris . The remaining four clusters could be differentiated both genotypically and phenotypically from each other and existing Rahnella species. Based on these results, we propose the description of four novel species: Rahnella perminowiae sp. nov. (type strain SL6 T =LMG 32257 T =DSM 112609 T ), Rahnella bonaserana sp. nov. (H11b T =LMG 32256 T =DSM 112610 T ), Rahnella rivi sp. nov. (FC061912-K T =LMG 32259 T =DSM 112611 T ) and Rahnella ecdela sp. nov. (FRB 231 T =LMG 32255 T =DSM 112612 T ).
... IAA plays an important role in the physiological processes of plants, such as cell division (Patten and Glick 2002;Rangarajan et al. 2003;Yang et al. 2009). Rahnella aquatilis, isolated from drinking water and the rhizosphere of different plants (Brenner et al. 1998;Berge et al. 1991), is one of the plant-growth-promoting rhizobacteria (PGPR) (Beazley et al. 2009;Valverde et al. 2011). The main purposes of this study were to investigate (1) the effects of R. aquatilis on the As accumulation of V. natans and (2) the role of R. aquatilis in resistance of V. natans to arsenic stress. ...
Vallisneria natans (Lour.) Hara is a suitable submerged plant for the phytoremediation of As-contaminated water. Rahnella aquatilis is one of the plant growth–promoting rhizobacteria. Influences of R. aquatilis on the arsenic accumulation and detoxification of V. natans were investigated. The results showed that As accumulation by V. natans could be significantly improved after R. aquatilis inoculated at the lower level of As (< 2 mg/L). At 0.5, 1, and 2 mg/L As levels, the As concentrations of V. natans with R. aquatilis were respectively 100.40%, 57.96%, and 22.62% higher than that of V. natans with no R. aquatilis. The concentration of As in V. natans was increased with the increasing the As concentration up to 1 mg/L, but it was decreased at 2 mg/L As. The correlation analysis showed that the As accumulated in the plant was positive correlated (R² = 0.977, p < 0.01) with indole-3-acetic acid (IAA) produced by R. aquatilis under different As levels. IAA may be the major factor affecting the As accumulation of V. natans. The results of malondialdehyde and superoxide dismutase, hydrogen peroxidase, and ascorbate peroxidase indicated that IAA produced by R. aquatilis had alleviated the arsenic stress on V. natans. The synthesis of IAA by R. aquatilis was related to the As levels. When the As was at 2 mg/L, the IAA that produced by R. aquatilis decreased and the promotion of R. aquatilis on As accumulation by V. natans reduced. However, R. aquatilis has a positive influence on the arsenic accumulation by V. natans at the lower As levels (< 2 mg/L), and it may be a potentially useful way to improve the removal of arsenic from contaminated water using submerged plants.
The present investigation aims to isolate nitrogen fixing endophytic bacteria from cereals crops and their potential role in plant growth promotion of wheat (Triticum aestivum L.) for sustainable growth. In the present investigation, endophytic bacteria were isolated from different cereal crops growing in the Divine Valley of Baru Sahib, Himachal Pradesh, India and isolates were screened for nitrogen fixation. The nitrogenase activity exhibiting bacterial isolates were further screened for other plant growth promoting traits including solubilization of phosphorus, potassium, and zinc; production of indole-3-acetic acid, siderophores, ammonia, hydrogen cyanide and extracellular enzyme. The potential nitrogen fixing strains were molecularly identified and evaluated for the growth promotion of wheat. A total of 304 putative endophytic bacterial isolates were isolated from wheat, oats, barley, and maize using selective and complex growth media. Among 304 putative endophytic bacteria, 8 isolates exhibits nitrogenase activity. On the basis of nitrogenase activity and other plant promoting traits, two efficient strains i.e. EU-E1ST3.1 and EU-A2RNfb were molecularly identified using 16S rRNA gene sequencing and found that these strains belongs to genera Rahnella. The wheat inoculated with two selected nitrogen-fixing endophytic bacterial strains showed considerable enhancement in total chlorophyll, nitrogen, Fe and Zn content over the un-inoculated control. In comparison of two selected nitrogen-fixing endophytic bacterial strains, Rahnella aquatilis EU-E1ST3.1 was found to enhance better growth and physiological parameters and it might be developed as biofertilizers to establish a sustainable agriculture system. In the present investigation, the isolated potential nitrogen fixing endophytic bacteria could be used as biofertilizer or bioinoculant for growth of diverse cereal crops growing in hilly region for agricultural sustainability.
The type strain Enterobacter agglomerans ATCC 27155 was examined for deoxyribonucleic acid (DNA) relatedness to 54 strains of the 'Erwinia herbicola-Enterobacter agglomerans complex', to 23 strains of other Erwinia and Enterobacter species, and to 50 reference strains of 49 different species in other genera of the family Enterobacteriaceae. The DNA-DNA hybridization values (at the optimal renaturation temperature; nitrocellulose filter method) showed that 24 strains were highly related to Enterobacter agglomerans ATCC 27155(T) (62 to 97% DNA relatedness) and formed a genotypic group provisionally called DNA hybridization group 27155. These strains were received as Enterobacter agglomerans (including strains of DNA hybridization groups V and XIII [D.J. Brenner, G.R. Fanning, J.K. Leete Knutson, A.G. Steigerwalt, and M.I. Krichevsky, Int. J. Syst. Bacteriol. 34:45-55, 1984]); as Erwinia herbicola, including the type strain NCPPB 2971; as Erwinia milletiae, including the type strain NCPPB 2519; and as yellow-pigmented Enterobacter strains. Numerical analysis of the protein electropherograms of these strains revealed the existence of seven protein electrophoretic groups, each showing a characteristic protein pattern. These seven groups separated at an infraspecific level and allowed assignment of 37 additional strains, receivecd as Enterobacter agglomerans, Erwinia herbicola, and Erwinia milletiae, to DNA hybridization group 27155. The resulting group hybridized below 55% DNA relatedness with all remaining phenotypic or genotypic groups of the 'Erwinia herbicola-Enterobacter agglomerans complex' and below 57% DNA relatedness with other Erwinia and Enterobacter species. DNA binding with 49 other species of the family Enterobacteriaceae was less than 38%. Since DNA hybridization group 27155 contains the type strains of Erwinia herbicola, Erwinia milletiae, and Enterobacter agglomerans, these species names are subjective synonyms, and the specific epithet agglomerans has priority. Further genotypic studies with several closely related genera are required for final placement of this species in a genus. The description of Enterobacter agglomerans, Erwinia herbicola, and Erwinia milletiae is emended.
Deoxyribonucleic acid (DNA)-DNA hybridization was performed with 10 strains belonging to the “Erwinia herbicola-Enterobacter agglomerans complex” by using the competition method on nitrocellulose filters. These strains exhibited more than 75% DNA binding to Erwinia herbicola ATCC 14589T (T = type strain) and constitute DNA hybridization group 14589 (including strains ATCC 14589T and CDC 1429-71 from DNA hybridization group III [D. J. Brenner, G. R. Fanning, J. K. Leete Knutson, A. G. Steigerwalt, and M. J. Krichevsky, Int. J. Syst. Bacteriol. 34:45-55, 1984]). The high level of genomic relatedness of these strains was confirmed by the similarities observed in their electrophoretic protein patterns. On the basis of our data, DNA hybridization group 14589 constitutes a discrete species within the family Enterobacteriaceae. Its closest relative is DNA hybridization group 27155 (41 to 53% DNA relatedness), which was previously defined and includes the type strains, among others, of Enterobacter agglomerans, Erwinia herbicola, and Erwinia milletiae (A. Beji, J. Mergaert, F. Gavini, D. Izard. K. Kersters, H. Leclerc, and J. De Ley, Int. J. Syst. Bacteriol. 38:77-88, 1988). We propose to unite DNA hybridization groups 14589 and 27155 in a single genus, Pantoea gen. nov. Pantoea agglomerans (Beijerinck 1888) comb. nov. is proposed to contain most strains of DNA hybridization group 27155 (including DNA hybridization group XIII of Brenner et al.), and its type strain is strain ATCC 27155 (= NCTC 9381 = LMG 1286). Pantoea dispersa sp. nov. is proposed to contain DNA hybridization group 14589, and its type strain is strain ATCC 14589 (= LMG 2603). Descriptions of the genus and its two species are given.
Acetylene-reducing bacteria isolated from the setts (stem cuttings used as seed pieces) and roots of two sugar cane varieties propagated aseptically from stem cuttings were identified using a computer-assisted scheme based on 75 biochemical tests. Because 106 to 108 acetylene-reducing bacteria per gram (fresh weight basis) were found in the roots, while 10 to 100 times fewer were present in the sett, we suggest that the root is the site of bacterial multiplication. Sterilization of the sett surface before planting or root sterilization at harvest reduced or completely removed acetylene-reducing bacteria and associated whole plant acetylene-reducing activity. This indicates that most of the active bacteria were on the sett and root exteriors. Setts did not exhibit acetylene-reducing activity until after emergence of the roots. Since shoot emergence was not necessary for acetylene-reducing activity, the extensive carbohydrate supply of the sett itself must have provided the carbon substrate for bacterial N2 fixation. The acetylene-reducing bacteria isolated were facultative anaerobes of the families Enterobacteriaceae and Bacillaceae. Klebsiella pneumoniae, Enterobacter cloacae, Erwinia herbicola, and Bacillus polymyxa were present inside the sett and the roots but E. herbicola was the dominant bacterium on the root exterior. No Beijerinckia spp. or Azotobacter spp. were found associated with the sett or the roots.
Soil samples from a number of tussock-grassland soils have been examined for the presence of non-symbiotic nitrogen-fixing micro-organisms and estimates made of the contribution by these organisms to the nitrogen status of tussock-grassland soils.Blue–green algae belonging to three genera (Anabaena, Nostoc, and Tolypothrix) and bacteria belonging to seven genera (and Rhodopseudomonas) were isolated, although of these only Clostridium species was found to be ubiquitous. Estimated numbers of these organisms in the soil were generally low.Acetylene-reduction assay of unamended soil samples indicated annual rates of nitrogen fixation equivalent to 0-3 kg N/ha.
A method for comparing the nitrogen-fixing activity of 40 rhizosphere bacteria was carried out using rice plantlets. Due to the large number of strains studied, the comparison was divided into different experiments. However, the same N2-fixing strain was used as a positive control. The comparison of nitrogenase activity (acetylene-reduction assay) of bacterial strains was conducted using the Dunnett's procedure. Using this method, out of the 17 different strains isolated from Bangladesh, Azospirillum lipoferum MRB16 was found to be the most efficient N2-fixing strain in association with rice cv. Nizershail. When a similar method was used to screen 23 N2-fixing strains from Egyptian soils in association with rice cv. Giza 171, A. brasilense NO40 was the most active isolate.
Antisera were prepared against three Rhizobium spp, (Cicer arietinum L.) strains H45, R18 and 46b4. Results obtained by the ELISA technique complement those obtained by immunofluorescence and revealed a broad serological diversity in the chickpea Rhizobium strains used. This serological diversity allowed us to use the immunofluorescence technique for competitiveness studies between an inoculated strain (H45) and native strains. Strain H45 formed all the nodules on plants cultivated on an acidic soil, 48 per cent of Montpellier soil and 11 per cent on Montelimar soil. The inoculum concentration was 5.2 107 bacteria per seed. On the Montpellier soil, the ratio of nodules formed by H45 increased with the inoculum concentration. The competitiveness of strain H45 against native strains of Montpellier soil is highly influenced by the host plant genotype. In each case, no cross reaction were observed between native strains of the soils studied and the antisera prepared against H45.
This study is an attempt to describe the dominant N2-fixing microflora associated with the roots of wetland rice. Rice cultivar Giza 171 was grown in a phytotron on two alluvial Egyptian soils for 8 days, a stage when the nitrogenase activity of undisturbed plants reached a level of 245 10–6 mol C2H4 h–1 g–1 dry weight of leaf. The roots and rhizosphere soils were then used for counting and isolating dominant diazotrophs. Counts and initial enrichment steps were carried out on a selective medium made of an axenic rice plantlet, the spermosphere model, incubated under 1 % acetylene. The counts were very high, exceeding 108 bacteria g–1 dry weight of rhizosphere soil. Enterobacteriaceae were dominant; most isolates were Enterobacter cloacae belonging to different biotypes in the two soils. Enterobacter agglomerans, Citrobacter freundii and Klebsiella planticola were also present as members of the dominant microflora. Azospirillum brasilense and Azospirillum lipoferum were present as well, but less abundant.