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Inoculation of nitrogen-free semi-solid media and monitoring the pellicle formed in the media. a . Veil like pellicle formed 2 days after inoculation. b . Surface/subsurface pellicle formed 7 days after inoculation. The black arrows in figures indicate the characteristic pellicle of the diazotrophic bacteria during growth in
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Background and aim: Nitrogen-fixing bacteria or diazotrophs have been isolated for many years using different formulations of N-free semi-solid media. However, the strategies used to isolate them, and the recipes of these media, are scattered through the published literature and in other sources that are more difficult to access and which are not a...
Contexts in source publication
Context 1
... most microaerobic diazotrophs the MPN method using N-free semi-solid media relies upon the appearance of a typical diazotrophic bacterial pellicle in the subsurface of the medium after incubation for 7 – 10 days at 30oC ( Fig. 3). Observation of the initial pellicle formation is usually possible 2 – 3 days after inoculation and growth must be then observed every subsequent day as some bacteria can grow very quickly. Counting can generally be performed after 5 – 7 days growth (Fig. 3). The characteristic bacterial pellicles in vials with the highest dilution are transferred to a fresh N-free semisolid medium, and after confirmation of bacterial growth, a loopfull of the new pellicle is streaked onto the corresponding solid semi-specific medium containing a trace amount of yeast extract (around 40 mg L − 1 ) to isolate the target bacterium based on the phenotypic characteristics of the colonies (Döbereiner 1995) (Fig. 4). A single purified colony is again checked in the same N-free semi-solid medium and the flasks that originally contained the characteristic pellicle are used for isolation of the bacteria and subsequent purification on potato agar medium (Baldani et al. 2000) (Fig. ...
Context 2
... most microaerobic diazotrophs the MPN method using N-free semi-solid media relies upon the appearance of a typical diazotrophic bacterial pellicle in the subsurface of the medium after incubation for 7 – 10 days at 30oC ( Fig. 3). Observation of the initial pellicle formation is usually possible 2 – 3 days after inoculation and growth must be then observed every subsequent day as some bacteria can grow very quickly. Counting can generally be performed after 5 – 7 days growth (Fig. 3). The characteristic bacterial pellicles in vials with the highest dilution are transferred to a fresh N-free semisolid medium, and after confirmation of bacterial growth, a loopfull of the new pellicle is streaked onto the corresponding solid semi-specific medium containing a trace amount of yeast extract (around 40 mg L − 1 ) to isolate the target bacterium based on the phenotypic characteristics of the colonies (Döbereiner 1995) (Fig. 4). A single purified colony is again checked in the same N-free semi-solid medium and the flasks that originally contained the characteristic pellicle are used for isolation of the bacteria and subsequent purification on potato agar medium (Baldani et al. 2000) (Fig. ...
Similar publications
Background and aims
Straw amendment can increase nitrogen fixation in paddy field, however, the efficiency of carbon sources with different biochemical properties to enhance N2 fixation and nitrogen fixation activity is still unclear.
Methods
A ¹⁵N2-labelling system was used in the field environment to determine biological nitrogen fixation (BNF)...
Citations
... The species classification of this strain was based on phenotypic characteristics, phylogenetic analysis and 16S rRNA G+C characterization (Gen Bank accession No: JF825470; ATCC BAA-2268) (Ismail and Dadrasnia, 2015). The strain was screened for plant growth-promoting features and found positive for N-fixing activity using the method of Baldani et al. (2014) and able to produce indole acetic acid (IAA) (18.5 ± 0.4 µg/mL) based on the method described by Gang et al. (2019). The B. salmalaya strain 139SI also gave a positive result for phosphate solubilization, as evaluated with the National Botanical Research Institute's Phosphate (NBRIP) plate culture (Mehta and Nautiyal, 2001) and siderophore production based on the chrome-azurol S approach (Louden et al., 2011). ...
Aims: Plant-microbe interaction in the rhizosphere significantly influences nutrient uptake efficiency. Thus, this research was aimed to investigate the potential of Bacillus salmalaya strain 139SI in increasing nutrient use efficiency through its synergistic effects with fertilizer application. Methodology and results: This research analyzed the effects of B. salmalaya strain 139SI inoculant, fertilizer and a combination of both on soil nutrients, vegetative growth, chlorophyll level, photosynthetic activities, nutrient uptake and nutrient use efficiency in oil palm seedlings for four months in a nursery setting. At the end of the research, the inoculation of B. salmalaya strain 139SI resulted in a significant increase in palm growth, chlorophyll level, photosynthetic activities, nutrient uptake and nutrient use efficiency compared to the untreated group. Soil nutrient analysis demonstrated that the inoculation of B. salmalaya strain 139SI led to a notable increase in available nitrogen within the rhizosphere soil. The findings of this research also indicated a noteworthy synergistic effect between the B. salmalaya strain 139SI inoculant and fertilizer. The most promising outcomes for plant growth performance and nutrient uptake were observed when the B. salmalaya strain 139SI inoculant was added to the fertilized palm. Conclusion, significance and impact of study: This research shows that B. salmalaya strain 139SI may work synergistically with fertilizer to enhance nutrient absorption and increase fertilizer usage efficiency. Integrating B. salmalaya into the nutrient management of oil palm seedlings can potentially reduce reliance on synthetic fertilizers, offering advantages to both farmers and the ecosystem.
... Diazotrophic (Gluconacetobacteria diazotrophicus) bacteria that fix nitrogen have recently been reported from the sugarcane plants. Since the endophyte can fix nitrogen and multiply, it has been hypothesised that it can meet the host plant's needs for nitrogen (Baldani et al 2014). ...
Biotechnology is one of the emerging fields that can add new and better application in a wide range of sectors like health care, service sector, agriculture, and processing industry to name some. This book will provide an excellent opportunity to focus on recent developments in the frontier areas of Biotechnology and establish new collaborations in these areas. The book will highlight multidisciplinary perspectives to interested biotechnologists, microbiologists, pharmaceutical experts, bioprocess engineers, agronomists, medical professionals, sustainability researchers and academicians. This technical publication will provide a platform for potential knowledge exhibition on recent trends, theories and practices in the field of Biotechnology.
... Bacterial isolates repeatedly selected on FLNFM were inoculated in another nitrogen-free FLNFM (Watanabe et al., 1979;Baldani et al., 2014) at pH 7.0 and incubated for 72 h at 30°C. Next, 100 μL of the bacterial isolates were added to gas chromatography vials (with cotton plugs) containing N-free semi-solid medium (0.385% agar) and incubated for 72 h at 30°C. ...
... The assay involved using a gas chromatograph (Agilent 6000 series) fitted with an HP-5 ms Capillary Column (50 m × 0.53 mm × 10 μm) at run time of 10 min and with an oven temperature of 150°C. Additionally, 100 μL from the same culture tested in the acetylene reduction assays was spread onto FLNFM agar (Watanabe et al., 1979;Baldani et al., 2014) and CFUs were recorded. ...
Introduction
Biological nitrogen fixation (BNF), an unparalleled metabolic novelty among living microorganisms on earth, globally contributes ~88-101 Tg N year⁻¹ to natural ecosystems, ~56% sourced from symbiotic BNF while ~22-45% derived from free-living nitrogen fixers (FLNF). The success of symbiotic BNF is largely dependent on its interaction with host-plant, however ubiquitous environmental heterotrophic FLNFs face many limitations in their immediate ecological niches to sustain unhindered BNF. The autotrophic FLNFs like cyanobacteria and oceanic heterotrophic diazotrophs have been well studied about their contrivances acclimated/adapted by these organisms to outwit the environmental constraints for functional diazotrophy. However, FLNF heterotrophs face more adversity in executing BNF under stressful estuarine/marine/aquatic habitats.
Methods
In this study a large-scale cultivation-dependent investigation was accomplished with 190 NCBI accessioned and 45 non-accessioned heterotrophic FLNF cultivable bacterial isolates (total 235) from halophilic estuarine intertidal mangrove niches of Indian Sundarbans, a Ramsar site and UNESCO proclaimed World Heritage Site. Assuming ~1% culturability of the microbial community, the respective niches were also studied for representing actual bacterial diversity via cultivation-independent next-generation sequencing of V3-V4 rRNA regions.
Results
Both the studies revealed a higher abundance of culturable Gammaproteobacteria followed by Firmicutes, the majority of 235 FLNFs studied belonging to these two classes. The FLNFs displayed comparable selection potential in media for free nitrogen fixers and iron-oxidizing bacteria, linking diazotrophy with iron oxidation, siderophore production, phosphorus solubilization, phosphorus uptake and accumulation as well as denitrification.
Discussion
This observation validated the hypothesis that under extreme estuarine mangrove niches, diazotrophs are naturally selected as a specialized multidimensional entity, to expedite BNF and survive. Earlier metagenome data from mangrove niches demonstrated a microbial metabolic coupling among C, N, P, S, and Fe cycling in mangrove sediments, as an adaptive trait, evident with the co-abundant respective functional genes, which corroborates our findings in cultivation mode for multiple interrelated metabolic potential facilitating BNF in a challenging intertidal mangrove environment.
... The reference strain R. pseudosolanacearum GMI1000 was also used in the present work (Prior et al. 2016;Safni et al. 2014;Salanoubat et al. 2002). G. diazotrophicus Pal 5 was grown in LGI-P medium (Baldani et al. 2014) containing (per liter): sucrose, 100.0 g; K 2 HPO 4 , 0.2 g; KH 2 PO 4 , 0.6 g; MgSO 4 ⋅7H 2 O, 0.2 g; CaCl 2 ⋅2H 2 O, 0.2 g; Na 2 MoO 4 ⋅H 2 O, 0.002 g; FeCl 3 ⋅6H 2 O, 0.01 g; pH adjusted to 5.5 at 30 °C and constant stirring of 200 rpm providing aerobic conditions. R. pseudosolanacearum growth in BG medium (Boucher et al. 1985) containing (per liter): Casein-Peptone, 10.0 g; Yeast Extract, 1.0 g; Casamino acids, 1.0 g. ...
Background and aims
This research investigates the biocontrol mechanisms of Gluconacetobacter diazotrophicus against the harmful bacterium Ralstonia pseudosolanacearum, causal agent of bacterial wilt disease. Given the ongoing need for effective control strategies, our focus lies in understanding the hormonal pathways, particularly the salicylic acid (SA) and jasmonic acid/ethylene (JA/Et) signaling pathways, involved in plant defense.
Methods
SA signalization deficiencies effects in plant-endophyte-pathogen interactions were observed through colonization ability, morpho-anatomical, physiological and disease severity parameters of sid2 (unable to produce SA via isochorismate synthase 1) and NahG (unable to accumulate SA) compared to wild-type Col0 plants treated with G. diazotrophicus, R. pseudosolanacearum or both bacteria. Additionally, pr1, pr5, myc2, pdf1.2 (associated with SA or JA/Et defense response), and fitness (linked to redox homeostasis) relative expression were analyzed in Col0.
Results
G. diazotrophicus increased root hairs in Col0 and NahG. This correlated with a higher endophytic colonization frequency and enhanced lignification and xylem expansion. However, chlorophyll content and bacterial counts indicated an endophyte-NahG imbalance. Disease index, R. pseudosolanacearum bacterial counts and H2O2 accumulation were lower in G. diazotrophicus inoculated Col0 and expression analysis revealed up-regulation of pr1 and myc2. In co-inoculated plants, R. pseudosolanacearum increased pdf1.2 levels by 15.2-times, which was significantly higher than in those inoculated solely with pathogenic bacteria. G. diazotrophicus managed to maintain fitness low levels even after R. pseudosolanacearum infection.
Conclusions
The interplay G. diazotrophicus-Arabidopsis, mediated by the expression of pr, myc2 and fitness primes for subsequent infection with R. pseudosolanacearum, triggering the expression of JA/Et pathway genes.
... The strains and plasmids used in this study are listed in Table 1. A. brasilense strains were grown at 28°C with shaking, in the minimal medium for A. brasilense (MMAB medium) supplemented with appropriate antibiotics (58). When the MMAB medium lacking a nitrogen source was used, cells were grown at 28°C without shaking to allow for microaerobic conditions that permit nitrogen fixation (59). When using solidified media, we used Noble agar because our laboratory has repeatedly observed that other sources of agar contain traces of nitrogen that inhibit the expression of nitrogen fixation genes. ...
Motile plant-associated bacteria use chemotaxis and dedicated chemoreceptors to navigate gradients in their surroundings and to colonize host plant surfaces. Here, we characterize a chemoreceptor that we named Tlp2 in the soil alphaproteobacterium Azospirillum brasilense. We show that the Tlp2 ligand-binding domain is related to the 4-helix bundle family and is conserved in chemoreceptors found in the genomes of many soil- and sediment-dwelling alphaproteobacteria. The promoter of tlp2 is regulated in an NtrC- and RpoN-dependent manner and is most upregulated under conditions of nitrogen fixation or in the presence of nitrate. Using fluorescently tagged Tlp2 (Tlp2-YFP), we show that this chemoreceptor is present in low abundance in chemotaxis-signaling clusters and is prone to degradation. We also obtained evidence that the presence of ammonium rapidly disrupts Tlp2-YFP localization. Behavioral experiments using a strain lacking Tlp2 and variants of Tlp2 lacking conserved arginine residues suggest that Tlp2 mediates chemotaxis in gradients of nitrate and nitrite, with the R159 residue being essential for Tlp2 function. We also provide evidence that Tlp2 is essential for root surface colonization of some plants (teff, red clover, and cowpea) but not others (wheat, sorghum, alfalfa, and pea). These results highlight the selective role of nitrate sensing and chemotaxis in plant root surface colonization and illustrate the relative contribution of chemoreceptors to chemotaxis and root surface colonization.
IMPORTANCE
Bacterial chemotaxis mediates host-microbe associations, including the association of beneficial bacteria with the roots of host plants. Dedicated chemoreceptors specify sensory preferences during chemotaxis. Here, we show that a chemoreceptor mediating chemotaxis to nitrate is important in the beneficial soil bacterium colonization of some but not all plant hosts tested. Nitrate is the preferred nitrogen source for plant nutrition, and plants sense and tightly control nitrate transport, resulting in varying nitrate uptake rates depending on the plant and its physiological state. Nitrate is thus a limiting nutrient in the rhizosphere. Chemotaxis and dedicated chemoreceptors for nitrate likely provide motile bacteria with a competitive advantage to access this nutrient in the rhizosphere.
... Sphingomonas was dominant in the early decay litters (9.26-16.83%). It can use all sorts of organic matters, including soluble and relatively complex types, to produce proteolytic or cellulolytic enzymes [35,36]. Some members of Methylobacterium were frequently identified as dominant taxa in the phyllosphere of various plants, where the bacteria of soil invade and participate in litter decomposition; this pattern was consistent with the bacterial succession during the decomposition of litters [37]. ...
In order to understand the role of microorganisms in litter decomposition and the nutrient cycle in volcanic forest ecosystems, the dominant forest species Larix gmelinii in the volcanic lava plateau of the Wudalianchi volcano was considered as the research object. We analyzed the response of bacterial community structure and diversity to litter decomposition for 1 year, with an in situ decomposition experimental design using litter bags and Illumina MiSeq high-throughput sequencing. The results showed that after 365 days, the litter quality residual rate of Larix gmelinii was 77.57%, and the litter N, P, C:N, C:P, and N:P showed significant differences during the decomposition period (p < 0.05). The phyla Cyanobacteria and the genus unclassified_o_Chloroplast were the most dominant groups in early decomposition (January and April). The phyla Proteobacteria, Actinobacteriota, and Acidobacteriota and the genera Massilia, Pseudomonas, and Sphingomona were higher in July and October. The microbial communities showed extremely significant differences during the decomposition period (p < 0.05), with PCoa, RDA, and litter QRR, C:P, and N as the main factors driving litter bacteria succession. Microbial functional prediction analysis showed that Chloroplasts were the major functional group in January and April. Achemoheterotrophy and aerobic chemoheterotrophy showed a significant decrease as litter decomposition progressed.
... The twelve strains isolated were inoculated at the centre of glucose nitrogen free mineral medium (G-NFMM) agar plates containing bromothymol blue solution (BTB) Appearance of green colour indicated that the isolate had nitrogen fixing activity. 7 The coloured zone index was computed on the third day for the isolates that produced blue colored zones on the agar plate. ...
Preparation of organic waste based bioformulation through solid state fermentation technology for application in the field offers an ecofriendly and viable alternative to sustainable food production. The present study deals with the potential of seven agrowastes as substrate carriers for the growth of indigenous strains of Pseudomonas fluorescens (Pf) and Bacillus subtilis (Bs) that stimulate plant growth. The cosubstrate (CS) with equal proportion of the seven agrowastes and both the bioinoculants (CSPfBs) proved as the best substrate carrier with highest population load of the bioinoculants at 45 days after storage (DAS). Thereafter the population declined steadily in all the bioformulations from 60 DAS to 90 DAS. However, CSPfBs gave the highest population load at 90 DAS. As individual substrate for both Pf and Bs, vermicompost performed best with a population load of 9.95 of Pf and 9.98 of Bs at 90 DAS. The bioformulations CSPfBs, VBs and VPf when applied in soil, gave the best yield in brinjal plants (Solanum melongena L. cv. Pusa Kranti) at 30 days after transplanting. Similar trends were also observed in the other yield parameters. The study indicates the scope of the prepared bioinoculant as a low cost eco friendly option for promotion of growth and yield of crops.
... The genus Nitrospirillum was proposed ten years ago by Lin et al. [1], when the species Azospirillum amazonense was transferred to this genus after a study based on available genomes. The former species had been described in the 1980s and contained a set of strains that possess distinct physiological characteristics from the other strains initially described as A. brasilense and A. lipoferum species [2]. A. amazonense was isolated and identified using nitrogenfree LGI semi-solid medium, derived from the pH and carbon source modifications of the original LG medium [3,4]. After the initial description of the Nitrospirillum genus, a new species, Nitrospirillum iridis, was subsequently proposed in references [5,6]. ...
... Catalase activity was assessed by flooding a colony with 3% (v/v) H 2 O 2 and observing the presence of bubbles. Differences in colony morphology were evaluated on LGI solid medium, and growth capacity was assessed on potato agar medium containing malic acid plus crystal sugar or only crystal sugar, final concentrations of 0.5% and 10%; incubation period was 10 days at 30 °C [4]. Tolerance to growth under salt conditions was evaluated by culturing the strains in liquid Dygs medium supplemented with 1 and 3% NaCl and monitoring the growth through medium turbidity for 5 days at 30 °C. ...
... These strains can thrive in a culture medium without fixed nitrogen due to the presence of nitrogenase; which enables the reduction of N 2 . The presence of a pellicle on the surface of the medium indicates the growth of these strains [4]. More detailed information on the isolation and growth conditions for these strains can be found at Baldani et al. [4]. ...
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 11140T = AM 18T = Y-2T = DSM 2788T = ATCC 35120T, BR 11142T = AM 14T = Y-1T = DSM 2787T = ATCC 35119T, 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 N2O 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 11140T (DSM 2788T, ATCC 35120T) designated as the type strain.
... Phosphorus solubilization was tested according to the method described by Ringuet et al. (2011) and Gupta and Pandey (2019). Potassium solubilization was done following the method described by Zhang and Kong (2014), and nitrogen fixation was tested according to Baldani et al. (2014). Endophytes were grown overnight in nutrient broth in falcon tubes at 30 °C. ...
Organic and natural sources of bio-stimulant have a great expectancy to boost green agriculture practices for sustainable, safe, and smart cultivation of crops. In that regard, beneficial endophytic bacteria have great potential. They have unique features in promoting plant growth by colonizing and establishing well in plant roots. In this study, endophytes isolated from the roots of moringa, neem, sesbania, and chilli were screened for crop’s growth-enhancing activities, such as phosphorus (P) solubilization, 1-aminocyclopropane-1-carboxylic-acid deaminase (ACC deaminase) production, and indole-acetic acid (IAA) production. The phosphorus solubilization, indole-acetic acid production, and ACC deaminase production values fall in the range of 55–88 ppm, 20–164 ppm, and 0.317–0.375 mM, respectively. Chilli seeds’ three highest vigor index (VI) values were attained by MR10 (12,457 VI), MR3 (9450 VI), and MR13 (8730 VI). MR13 showed the highest seed germination energy (221%), followed by MR1 (178%) and MR3 (156%). The promising endophytes were tested on chilli seedlings as single and mixed inoculum treatments to study the efficiency of root colonization. Mixed cultures containing CKR8 and MR13 exhibited the highest seedling height (17.0 cm), followed by MR13, MR10, and MR13 (16.8 cm) compared to the control (12.6 cm). A single culture of MR10 (109.0 g and 13.53 cm²) and a mixed culture of MR10 and MR13 (100.0 g and 13.09 cm²) showed the maximum root length and surface area, respectively. The highest relative chlorophyll content was recorded by MR10 and MR13 (40.3 SPAD value), followed by MR13, MR3, and CKR8 (36.8 SPAD value). The non-native endophytic bacteria, MR13, Streptomyces panaciradicis (GenBank accession no. OM001090), and MR3, Bacillus subtilis (GenBank accession no. OM714810), could colonize the roots and improve the growth of chilli at the seedling growth stage.
Graphical Abstract
... For the bacterial inoculum, a pure culture of Azospirillum argentinense REC3 (dos Santos Ferreira et al., 2022) was grown in NFb liquid medium (Baldani et al., 2014), supplemented with 1% NH4Cl for 24 h at 30°C and 120 rpm. Cells were then centrifuged at 2000 x g for 10 min and washed twice with sterile bi-distilled water at pH 7.0 to remove culture medium residues that might interfere with the assays; cells were suspended in sterile bi-distilled water and the concentration for seed inoculation was ~10 6 CFU.mL -1 (OD560 nm 0.2). ...
Root research often uses rhizotron systems where their main characteristic is to possess a transparent wall allowing the in situ observation of plants’ root system. Rhizotrons can vary in size and be filled with different substrates based on soil, sand and other components. Here, a simple and low cost rhizotron system without soil substrate was developed, allowing the observation of wheat (Triticum aestivum L.) root system treated with bioproducts. To show the effectiveness of the rhizotrons, hydrated wheat seeds were treated with plant growth-promoting bioproducts such as Azospirillum argentinense REC3 (106 CFU.ml-1), flagellin AzFlap (200 nM), or strawberry hydroalcoholic extract (0.01 mg.mL-1). This rhizotron system allowed to non-destructively evaluate the roots (length, density and root hair proliferation), where the values of the bioproducts surpassed the control.
