Nitrogen-free Medium Research Articles

Symbiodiniaceae and Ruegeria sp. Co-Cultivation to Enhance Nutrient Exchanges in Coral Holobiont.

The symbiotic relationship between corals and their associated microorganisms is crucial for the health of coral reef eco-environmental systems. Recently, there has been a growing interest in unraveling how the manipulation of symbiont nutrient cycling affects the stress tolerance in the holobiont of coral reefs. However, most studies have primarily focused on coral-Symbiodiniaceae-bacterial interactions as a whole, neglecting the interactions between Symbiodiniaceae and bacteria, which remain largely unexplored. In this study, we proposed a hypothesis that there exists an inner symbiotic loop of Symbiodiniaceae and bacteria within the coral symbiotic loop. We conducted experiments to demonstrate how metabolic exchanges between Symbiodiniaceae and bacteria facilitate the nutritional supply necessary for cellular growth. It was seen that the beneficial bacterium, Ruegeria sp., supplied a nitrogen source to the Symbiodiniaceae strain Durusdinium sp., allowing this dinoflagellate to thrive in a nitrogen-free medium. The Ruegeria sp.-Durusdinium sp. interaction was confirmed through 15N-stable isotope probing-single cell Raman spectroscopy, in which 15N infiltrated into the bacterial cells for intracellular metabolism, and eventually the labeled nitrogen source was traced within the macromolecules of Symbiodiniaceae cells. The investigation into Symbiodiniaceae loop interactions validates our hypothesis and contributes to a comprehensive understanding of the intricate coral holobiont. These findings have the potential to enhance the health of coral reefs in the face of global climate change.

Culturable Diversity of Endophytic and Rhizoplane Colonizing Bacteria of Indian Mustard (Brassica juncea (L.) Czern & Coss), Affected by Soil Types and Assessment of Plant Growth Promoting Attributes

Microbial community in the rhizosphere of plant significantly influences growth and development of a plant. As the soil vary in microbial composition due to interaction of soil with plant roots and its exudates, the role of soil in shaping microbial diversity of a crop rhizosphere is least studied. In present study, Mustard (Brassica juncea cv Pusa Mehak) was grown in three different soil viz. Vertisols, Inceptisols and Alfisols, and the bacteria were isolated form root tissues and rhizoplane to study their plant growth promoting (PGP) attributes and diversity using BOX-Polymerase chain reaction (PCR) fingerprinting technique, as influenced by soil types. Form threes soil orders, total 37 isolates were obtained of which, 19 could grow on nitrogen free medium, 6 solubilized P from tricalcium phosphate, 2 isolate solubilized potassium from glauconite and 8 had zinc solubilizing ability with the highest zinc solubilization index of 3.84 by isolate VRB-7. Based on the fingerprint analysis, differences occurred in the endophytic bacteria of same host grown in different soil types suggesting influence of soil types in colonization of microbes in rhizoplane and endorhizosphere. The common organisms obtained from host grown in different soil types indicating host genotype driven assembly of bacteria. Similarity in the isolates on rhizoplane and endorhizosphere indicated endophytic colonization of the isolates capable of firmly adhering to the rhizoplane. The findings of this study will help in formulation of crop specific bioinoculant for specific soil types and edaphic properties.

Description of Microbacterium dauci sp. nov., a plant growth hormone indoleacetic acid-producing and nitrogen-fixing bacterium isolated from carrot rhizosphere soil.

A plant growth hormone indoleacetic acid-producing strain LX3-4T was isolated from a carrot rhizosphere soil sample collected in Shandong Province, China. It is Gram-stain-positive, non-motile, and has irregular short rod-shaped cells. LX3-4T shared high 16S rRNA gene sequence identity with Microbacterium oleivorans DSM 16091T (99.4%), M. testaceum NBRC 12675T (98.6%), M. marinum DSM 24947T (98.5%), M. resistens NBRC 103078T (98.4%), and M. paraoxydans NBRC 103076T (98.3%). Phylogenetic analysis based on the concatenated gene sequences of 16S rRNA gene, housekeeping genes gryB and rpoB also showed the distinction between strain LX3-4T and other Microbacterium species. Furthermore, analysis of the average nucleotide identities (ANI), the average amino acid identity (AAI), and the digital DNA-DNA hybridization (dDDH) values between strain LX3-4T and its relatives revealed that strain LX3-4T represents a distinct species. The genomic DNA G + C content of the strain is 69.5%. It can grow at 25-37°C (optimum 37°C), pH 5.0-10.0 (optimum pH 6.0-8.0), and the range of NaCl concentration is 0-7% (w/v) (optimum 1-5%). The colonies on agar plates are smooth, translucent, and pale yellow. The main cellular fatty acids of strain LX3-4T are anteiso-C15:0, anteiso-C17:0, and iso-C16:0. The predominant respiratory quinones are MK-12 and MK-11. Diphosphatidylglycerol, phosphatidylglycerol, an unidentified glycolipid, and an unidentified phosphoglycolipid are major polar lipids. The cell-wall sugar of strain LX3-4T is glucose. The cell-wall peptidoglycan contains glycine, alanine, lysine, and glutamic acid. In addition, this strain carries nitrogen fixation genes and can grow in nitrogen-free medium. Based on the polyphasic data, strain LX3-4T represents a novel species of the genus Microbacterium, for which the name Microbacterium dauci sp. nov. is proposed with strain LX3-4T (= CCTCC AB 2023103T = LMG 33159T) designated as the type strain.

Restoration of the Functional nif Gene Cluster by Complex Recombination Events during Heterocyst Development in the Nitrogen-Fixing Cyanobacterium Calothrix sp. NIES-4101.

In the genome of the heterocystous cyanobacterium Calothrix sp. NIES-4101 (NIES-4101), the four genes essential for nitrogen fixation (nifB, nifH, nifD and nifK) are highly fragmented into 13 parts in a 350-kb chromosomal region, and four of these parts are encoded in the reverse strand. Such a complex fragmentation feature makes it difficult to restore the intact nifBHDK genes by the excision mechanism found in the nifD gene of the Anabaena sp. PCC 7120 heterocyst. To examine the nitrogen-fixing ability of NIES-4101, we confirmed that NIES-4101 grew well on a combined nitrogen-free medium and showed high nitrogenase activity, which strongly suggested that the complete nifBHDK genes are restored by a complex recombination process in heterocysts. Next, we resequenced the genome prepared from cells grown under nitrogen-fixing conditions. Two contigs covering the complete nifHDK and nifB genes were found by de novo assembly of the sequencing reads. In addition, the DNA fragments covering the nifBHDK operon were successfully amplified by PCR. We propose that the process of nifBHDK restoration occurs as follows. First, the nifD-nifK genes are restored by four excision events. Then, the complete nifH and nifB genes are restored by two excision events followed by two successive inversion events between the inverted repeat sequences and one excision event, forming the functional nif gene cluster, nifB-fdxN-nifS-nifU-nifH-nifD-nifK. All genes coding recombinases responsible for these nine recombination events are located close to the terminal repeat sequences. The restoration of the nifBHDK genes in NIES-4101 is the most complex genome reorganization reported in heterocystous cyanobacteria.

Pontiella agarivorans sp. nov., a novel marine anaerobic bacterium capable of degrading macroalgal polysaccharides and fixing nitrogen.

Marine macroalgae produce abundant and diverse polysaccharides, which contribute substantially to the organic matter exported to the deep ocean. Microbial degradation of these polysaccharides plays an important role in the turnover of macroalgal biomass. Various members of the Planctomycetes-Verrucomicrobia-Chlamydia (PVC) superphylum are degraders of polysaccharides in widespread anoxic environments. In this study, we isolated a novel anaerobic bacterial strain NLcol2T from microbial mats on the surface of marine sediments offshore Santa Barbara, CA, USA. Based on 16S ribosomal RNA (rRNA) gene and phylogenomic analyses, strain NLcol2T represents a novel species within the Pontiella genus in the Kiritimatiellota phylum (within the PVC superphylum). Strain NLcol2T is able to utilize various monosaccharides, disaccharides, and macroalgal polysaccharides such as agar and ɩ-carrageenan. A near-complete genome also revealed an extensive metabolic capacity for anaerobic degradation of sulfated polysaccharides, as evidenced by 202 carbohydrate-active enzymes (CAZymes) and 165 sulfatases. Additionally, its ability of nitrogen fixation was confirmed by nitrogenase activity detected during growth on nitrogen-free medium, and the presence of nitrogenases (nifDKH) encoded in the genome. Based on the physiological and genomic analyses, this strain represents a new species of bacteria that may play an important role in the degradation of macroalgal polysaccharides and with relevance to the biogeochemical cycling of carbon, sulfur, and nitrogen in marine environments. Strain NLcol2T (= DSM 113125T = MCCC 1K08672T) is proposed to be the type strain of a novel species in the Pontiella genus, and the name Pontiella agarivorans sp. nov. is proposed.IMPORTANCEGrowth and intentional burial of marine macroalgae is being considered as a carbon dioxide reduction strategy but elicits concerns as to the fate and impacts of this macroalgal carbon in the ocean. Diverse heterotrophic microbial communities in the ocean specialize in these complex polymers such as carrageenan and fucoidan, for example, members of the Kiritimatiellota phylum. However, only four type strains within the phylum have been cultivated and characterized to date, and there is limited knowledge about the metabolic capabilities and functional roles of related organisms in the environment. The new isolate strain NLcol2T expands the known substrate range of this phylum and further reveals the ability to fix nitrogen during anaerobic growth on macroalgal polysaccharides, thereby informing the issue of macroalgal carbon disposal.

Dynamic nitrogen fixation in an aerobic endophyte of Populus.

Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte-poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only ~11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through 15N2 enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant-endophyte interactions.

Plant growth promoting potential of Arthrobacter spp., an early colonizer on the volcanic deposits of Mt. Merapi, Indonesia, as a restorative agent for degraded land

Exploration of novel and effective plant growth-promoting bacteria (PGPB) from unexplored environments is an intriguing option for restoring degraded land in Indonesia caused by mining activity. Due to their ability to develop in the absence of carbon, PGPB derived from volcanic environments may be a sustainable alternative. Previous research on the volcanic deposits of Mt. Merapi, Indonesia and Miyake-Jima, Japan, revealed that Arthrobacter spp. dominated the pioneer bacterial communities. Consequently, this research aimed to investigate the biochemical and physiological characteristics of volcanic Arthrobacter groups and assess their potential as PGPB. Using the conventional API system (20NE, 50CH, Zym), biochemical characteristics were evaluated. In vitro evaluations of plant growth-promoting activities such as siderophore production, phosphate solubilization, growth in media devoid of nitrogen, heavy metal resistance and weathering activity were conducted. The results showed broad substrate utilization and enzyme activity profiles of the Mt. Merapi isolates (BRU 37, BRU I.13, and BRU I.45). During 15 days of incubation on a minimal medium containing potassium feldspar, glucose, and NH4Cl, all of the isolates grew and produced acids. In nitrogen-free media, all of the isolates grew. These isolates showed positive reaction for the phosphate solubilization assay using Pikovskaya plates. After one day of incubation, all isolates produced yellow zones on the blue-green pigmented media (CAS-overlay media) and were therefore classified as potent siderophore producers. The heavy metal resistance assay showed that BRU37 grew in the heavy metal media. These results indicate the potential of selected indigenous Mt. Merapi Arthrobacter to enhance plant growth. Further characterization of plant growth-promoting traits is now in progress.

Eksplorasi Potensi Bacillus spp. sebagai Bakteri Pemacu Pertumbuhan Tanaman di Hutan Primer Resort Kembang Kuning

Kembang Kuning resort is one of the Mount Rinjani National Park Resorts that have high biodiversity, so it has the potential for plant growth-promoting bacteria. Bacillus sp. is one species of bacteria that has great potential as a plant growth-promoting bacteria. The purpose of this study was to isolate and determine the ability of phosphate solubilization, nitrogen-fixing and anti-fungal activity of Bacillus spp. isolated from the Primary Forest of Kembang Kuning Resort, Gunung Rinjani National Park. This research is an exploratory study consisting of the isolation of Bacillus sp., phosphate solubilization test with spot inoculation method on Pikovskaya Agar media, qualitative nitrogen fixing test using Jensen's Nitrogen - Free media, and anti-fungal activity test against Fusarium sp. with dual culture method. There were 21 isolates of the genus Bacillus successfully isolated in this study. The phosphate solubilization test showed that 16 bacterial isolates could solubilize phosphate, where the highest phosphate solubilization index was produced by isolate P3.1 namely 0,52. A total of one isolate, namely isolate P3.1, can fix nitrogen on Jensen's Nitrogen-Free medium. In the anti-fungal test against Fusarium sp., three bacterial isolates were obtained that were able to inhibit the growth of Fusarium sp., where the highest antagonism was produced by isolate P2.8 which was 46,66%. Bacillus spp. isolated from Kembang Kuning Resort has potential as PGPB in terms of crop protection.

Properties of Potential Plant-Growth-Promoting Bacteria and Their Effect on Wheat Growth Promotion (Triticum aestivum) and Soil Characteristics

Plant-growth-promoting bacteria are an important economic and environmental resource as biofertilizers that can stimulate plant growth and improve agricultural yields. In this study, potential plant growth-promoting bacteria were isolated from soil samples collected in Russia. Strains that manifested active growth on a nitrogen-free medium, the Pikovskaya medium (with insoluble phosphates) and CAS (Chrome Azurol S) agar, were selected for the study. All bacterial isolates were identified by 16S rRNA gene sequencing analysis. Seventeen bacterial isolates of different species were purified and quantified for their ability to grow on nitrogen-free media; dissolve phosphate; and produce ammonium, indole-3-acetic acid, siderophores, and antifungal activity. Principal component analysis identified three groups of strains: one with the maximum signs of providing “plant nutrition”; one with signs of “antimicrobial activity”; and a group “without outstanding signs”. All 17 strains were involved in experiments involving growing inoculated wheat seeds (Triticum aestivum) in pots under natural environmental conditions, and were assessed by their effect on the wheat growth and yield as well as on the chemical composition of the soil. For the “plant nutrition” group, regression analysis revealed a connection between indicators of plant growth, ear length, and ammonium accumulation in the soil. However, in other groups, there were also strains showing a positive effect on plant growth, which suggests the necessity of involving additional factors to predict the ability of strains to affect plants when screened in the laboratory.

The Comparison of Tolerance Capability of Nitrogen-Fixing Bacteria to Chemical and Biological Pesticides

Due to concerns about the adverse effects of synthetic pesticides on the environment and human health, there is a growing preference for environmentally friendly and safe pesticides. However, hard evidence to verify their low-risk characteristics is scarce. This work attempted to compare the effects of a chemical pesticide (alpha-cypermethrin) and a biopesticide (emamectin benzoate) on the growth of indigenous nitrogen-fixing bacteria. These bacterial strains were isolated from the rhizosphere of bean plant samples obtained from farmers? fields where these two pesticides were sprayed on bean crops. The results revealed that at the field-recommended concentration of both pesticides, the growth of the isolates in both nutrient-rich and nitrogen-free media remained largely unaffected. However, the bacteria?s absolute growth inhibition was recorded as the pesticide concentration increased. When the Tryptic Soy Broth medium was supplemented with a pesticide concentration 10 times higher than the recommended dose, up to 85.7% of the isolated strains were completely inhibited in growth when exposed to alpha-cypermethrin. In contrast, only 17.9% of bacterial strains were completely inhibited when exposed to emamectin benzoate. In a nitrogen-free medium, the proportion of inhibited bacteria was absolute when the medium was supplemented with alpha-cypermethrin and emamectin benzoate at a dosage 10 times higher than the recommended concentration, which reached 92.9% and 28.6%, respectively. The strains that exhibited higher tolerance to both pesticides were identified as Niallia nealsonii ND15 and Rhizobium subbaraonis ND27. Our findings emphasize the importance of minimizing pesticide accumulation in the soil to protect the viability of soil-beneficial bacteria and ensure soil quality for safe and sustainable crop production.. KEYWORDS :Alpha-cypermethrin, Emamectin benzoate, N-fixing bacteria, Pesticides tolerance, Toxicity assessment

Groundnut harbours non-nodulating non-rhizobial plant growth-promoting bacterial endophytes.

The present study was carried out to assess the growth-promoting ability of non-rhizobial endophytes in groundnut (Arachis hypogaea). Thirteen endophytic bacteria with different morphologies were isolated from the root and nodules of groundnut. These isolates significantly enhanced the growth of groundnut in sterilised vermiculite, though the isolates were unable to nodulate the host plant. The endophytic nature of these isolates was confirmed by their re-isolation from the sterilised and macerated roots of the plants. The isolates exhibited in vitro tricalcium phosphate and zinc solubilization, production of siderophores, auxins and ammonia as well as growth on different nitrogen-free media. The phosphate solubilization and auxin production varied from 50 to 196 and 17 to 71µg/ml, respectively by the isolates. Based on phenotypic tests and 16S rRNA gene sequencing, four potential strains were identified as Klebsiella sp. R3, Pseudomonas putida R6, Klebsiella oxytoca GRE5 and Pseudomonas proteolytica GRE6. A significant increase in plant growth, chlorophyll content, nodule count and shoot nutrient content of groundnut was observed with these bacterial inoculations over the uninoculated control in greenhouse. The bacterial treatments resulted in increased N, P and K content in the shoot up to 87, 96 and 44%, respectively, over the control. Physico-chemical properties and available nutrient content of soil were also improved on bacterial inoculations. The results indicated that groundnut harbours beneficial non-rhizobial bacterial endophytes with the potential to be used as microbial inoculants in groundnut. Klebsiella oxytoca as a non-nodulating nodule endophyte of groundnut is reported for the first time.

Methylobacterium spp. mitigation of UV stress in mung bean (Vigna radiata L.).

Methylotrophs are a diverse group of bacteria that abundantly colonize the phyllosphere and have great potential to withstand UV irradiation because of their pigmented nature and ability to promote plant growth through various mechanisms. The present study investigated the effects of UVB radiation on plant growth-promoting (PGP) properties of methylotrophic bacteria and the growth of Vigna radiata L. A total of 55 methylotrophic bacteria were isolated from desert plants, and 15 methylotrophs were resistant to UVB radiation for 4h. All UVB-resistant methylotrophs possess a methyldehydrogenase gene. Identification based on 16S rRNA gene sequencing revealed that all 15 UVB-resistant methylotrophs belonged to the genera Methylorubrum (07), Methylobacterium (07), and Rhodococcus (01). Screening of methylotrophs for PGP activity in the presence and absence of UVB radiation revealed that all isolates showed ACC deaminase activity and growth on a nitrogen-free medium. Furthermore, the production of IAA-like substances ranged from 8.62 to 85.76µg/mL, siderophore production increased from 3.47 to 65.75% compared to the control. Seed germination assay with V. radiata L. (mung bean) exposed to UVB radiation revealed that methylotrophs improved seed germination, root length, and shoot length compared to the control. The present findings revealed that the isolates SD3, SD2, KD1, KD5, UK1, and UK3 reduced the deleterious effects of UVB radiation on mung bean plants and can be used to protect seedlings from UVB radiation for sustainable agriculture.

Protocol: an improved method for inducing sporophyte generation in the model moss Physcomitrium patens under nitrogen starvation

BackgroundLand plants exhibit a haplodiplontic life cycle, whereby multicellular bodies develop in both the haploid and diploid generations. The early-diverging land plants, known as bryophytes, have a haploid-dominant life cycle, in which a short-lived multicellular body in the diploid generation, known as the sporophyte, develops on the maternal haploid gametophyte tissues. The moss Physcomitrium (Physcomitrella) patens has become one of the most powerful model systems in evolutionary plant developmental studies. To induce diploid sporophytes of P. patens, several protocols are implemented. One of the conventional approaches is to grow approximately one-month-old gametophores for another month on Jiffy-7 pellets made from the peat moss that is difficult to fully sterilize. A more efficient method to obtain all tissues throughout the life cycle should accelerate studies of P. patens.ResultsHere, we investigated the effect of nitrogen conditions on the growth and development of P. patens. We provide an improved protocol for the sporophyte induction of P. patens using a BCD-based solid culture medium without Jiffy-7 pellets, based on the finding that the formation of gametangia and subsequent sporophytes is promoted by nitrogen-free growth conditions. The protocol consists of two steps; first, culture the protonemata and gametophores on nitrogen-rich medium under continuous light at 25 °C, and then transfer the gametophores onto nitrogen-free medium under short-day and at 15 °C for sporophyte induction. The protocol enables to shorten the induction period and reduce the culture space.ConclusionsOur more efficient and shortened protocol for inducing the formation of sporophytes will contribute to future studies into the fertilization or the diploid sporophyte generation of P. patens.

Plant growth promotion of radish by rhizosphere dwelling bacteria

The present study was carried out with the aim to develop a plant growth-promoting rhizobacterial inoculant for improving the growth of radish. A total of 20 rhizobacteria were isolated and then tested for multifarious plant growth-promoting traits. The isolates exhibited tricalcium phosphate and zinc solubilization, production of siderophores, auxins, ammonia and growth on different nitrogen free medium. Out of 20 isolates, two potential bacterial isolates (RM3 and RL3) were selected on the basis of PGP traits for evaluation of their potential to promote growth of radish under pot conditions. In green house experiment, these isolates when used as consortium showed a significant improvement in soil enzymatic activities (viz. dehydrogenase: 12.99 µg TPF formed h-1 g of soil-1, alkaline phosphatase: 11.94 µg PNP formed h-1 g of soil-1 and urease: 401.65 µg urea hydrolyzed formed h-1g of soil-1) and plant growth parameters (shoot length: 38.76 cm, leaf area: 267cm2, fresh leaf weight:106.05 g, dry leaf weight:1.23 g, number of leaves per plant: 23, fresh root weight:111.69 g, dry root weight: 2.06 g, root length: 34.04 cm and root diameter: 3.33 cm) relative to control as well as other bioinoculants. These bacteria were tentatively identified as Bacillus (RM3) and Pseudomonas (RL3) on the basis of biochemical tests.

Taxonomic and functional characteristics of xerotolerant culturable bacterial community of Negev desert soil

Despite the significant expansion of the geography of soil microorganisms studies in various, including extreme natural environments, research on the biodiversity and metabolic activity of soil microbial communities under conditions of moisture deficiency are few. Biodiversity of culturable bacteria isolated from the surface horizon of the Negev desert soil (Aridic Calcisol) after pre-incubation of soil samples under the conditions of low water availability has been studied. An increase in the diversity of culturable bacteria after pre-incubation was revealed in comparison with the community isolated from the native soil. From the native and pre-incubated samples, 153 phenotypically unique pure cultures of bacteria from 22 genera belonging to the phyla Actinomycetota, Pseudomonadota, Bacillota, and Bacteroidota were isolated and identified. The taxonomic diversity of culturable bacteria isolated from the sample pre-incubated at a water activity (Aw) of 0.90 was two times higher than that obtained from the native soil. The strain of the genus Pedobacter has been identified, which may be a representative of a previously undescribed bacterial species. Bacteria of the genera Aerococcus, Bacillus, Brevibacterium, Staphylococcus, and Stenotrophomonas capable of growing at Aw 0.91 were found. One strain of the genus Microlunatus has been identified that is capable of growing on a nitrogen-free medium, exhibiting amylase and protease activity, and capable to growth on a medium with Aw down to 0.96. The obtained results confirm the hypothesis that arid ecosystems, in particular, desert soils and sediments, are a depository of a previously unexplored taxonomic diversity of bacteria with unique physiological properties that are promising for study and potential implementation in biotechnological processes.

Оценка ростостимулирующих свойств ризобактерий Bacillus sp. и их влияние на морфофизиологические характеристики рапса

Abstract. The use of biofertilizers based on plant growth promoting (PGP) bacteria is a promising direction in agri-biotechnology. The purpose was to evaluate the PGP-attributes of Bacillus sp. and to reveal the morphophysiological features of rapeseed (Brassica napus L.) when inoculated with these rhizobacteria. Methods. The ability of Bacillus sp. strain TO15c isolated from the rhizosphere of Taraxacum officinale on Zack's nitrogen-free medium to produce indol-3-acetic acid (IAA) and phosphates was studied. In pot-scale experiments, the assessment of changes in the morphophysiological characteristics of rapeseed upon inoculation with rhizobacteria both in the absence and in the presence of nitrogen fertilizer was performed. At the end of 100-day vegetation, shoot length, total leaf area, fresh biomass, content of macronutrients and photosynthetic pigments were determined. Results. The ability of TO15c to produce IAA (up to 26 mg/L) and solubilize phosphate (up to 60 mg/L) has been proven. Soil inoculation with rhizobacteria in the presence of ammonium nitrate led to the increase in shoot length by 24 % and total leaf area by 16 %. The aboveground fresh biomass increased by 1.5 times, the underground by 2.5 times, and the content of macronutrients improved. The rise in the photosynthetic pigment content (1.5 times on average) was also noted when inoculated with TO15c. The maximum effect was achieved with the rhizobacteria and nitrogen fertilizer combined application. Scientific novelty. The effectiveness of Bacillus sp. TO15c on rapeseed increased in the presence of nitrogen fertilizer, despite the fact that these PGPR were able to fix atmospheric nitrogen.

Enhancing the ability of rice to adapt and grow under saline stress using selected halotolerant rhizobacterial nitrogen fixer

Abstract Salinity stress has become the major devastating constraint for rice growth. Halotolerant rhizobacterial nitrogen fixer (HRNF) was investigated for increasing the nitrogenase activity (NA), organic acid (OA), gibberellic acid (GA), and indole 3-acetic acid (IAA) productions, seedling growth, and rice yield. Six N fixers were isolated using Ashby’s (Ab1, Ab2, and Ab3) and Okon’s media (Az1, Az2, and Az3). Furthermore, bioassay was carried out using rice seedling grown on nitrogen-free medium. The Ab3 and Az2 isolates were selected and biomolecularly identified as Pseudomonas stutzeri and Klebsiella pneumoniae, respectively. These selected bacteria were used as active ingredients for Halotolerant rhizobacterial inoculant (HRI) dosage trials (0, 500, 1,000, and 1,500 g ha−1) on simple pot experiments. The Az group isolates had 3–5 times higher ability in fixing N and producing OA, GA, and IAA than the Ab group isolates. Furthermore, N-uptake, number of panicles, filled grain, and the rice yield of HRI treated pots were significantly increased. Application of 1,000–1,500 g HRI ha−1 had resulted in a significant increase in the yield of rice grain (26.10–28.27 g plant−1 or 15.4–25.09%) which was higher than the control. This result concludes that HRI could contribute in enhancing the ability of rice to adapt and grow under saline stress.

The effect of low temperature and nitrogen starvation on the morphological and physiological characteristics of two strains of green microalgae of the genus <i>Lobosphaera</i> sp.

The effect of nitrogen starvation and, for the first time, low temperature, as well as their simultaneouseffect, on the physiology and ultrastructure of cells of microalgae of the genus Lobosphaera (Chlorophyta, Trebouxiophyceae) was studied. Nitrogen deficiency in both strains led to a decrease in the content ofchlorophyll by three times and an increase in the proportion of carotenoids by two times. A decrease in thecontent of both chlorophyll and carotenoids was observed at +10C. The simultaneous effect of two factorsresulted in a threefold decrease in the chlorophyll content in NAMSU 924/2 and a sixfold decrease inNAMSU (CALU) 1497; the proportion of carotenoids in both strains decreased by 1.52 times. Data onultrastructural changes in cells of microalgae of the genus Lobosphaera under the influence of stress factorshave been obtained. A similar nature of the response in both strains to stress conditions was noted. Nitrogendeficiency led to the accumulation of numerous lipid droplets in the cytoplasm of cells along the cell wall. Long-term incubation on a nitrogen-free medium led to the filling of the entire volume of cells with lipiddroplets, disassembly of the membrane system of chloroplasts, that reduction in sizeand being locatedbetween densely lying lipid droplets. At low temperatures, the number of thylakoids decreased, while theinterthylakoid space and the size of chloroplasts increased. With simultaneous exposure to nitrogen starvation and low temperature, numerous lipid droplets accumulated, the number of thylakoids decreased, the interthylakoid space and the size of the chloroplast increased, which was noted under separate exposure to stress factors. The pyrenoid in both strains did not undergo significant changes in all cases.

A Novel Nitrogen-Fixing Bacterium Raoultella electrica Isolated from the Midgut of the Leafhopper Recilia dorsalis.

Nitrogen is a crucial element for the growth and development of insects, but herbivorous insects often suffer from nitrogen nutrition deficiencies in their diets. Some symbiotic microorganisms can provide insect hosts with nitrogen nutrition through nitrogen fixation. Extensive research has clearly demonstrated the process of nitrogen fixation by symbiotic microorganisms in termites, while evidence supporting the occurrence and significance of nitrogen fixation in the diets of the Hemiptera is less conclusive. In this study, we isolated a strain of R. electrica from the digestive tract of a leafhopper, R. dorsalis, and found that it had nitrogen-fixing capabilities. Fluorescence in situ hybridization results showed that it was located in the gut of the leafhopper. Genome sequencing revealed that R. electrica possessed all the genes required for nitrogen fixation. We further evaluated the growth rate of R. electrica in nitrogen-containing and nitrogen-free media and measured its nitrogenase activity through an acetylene reduction assay. The findings of these studies could shed light on how gut microbes contribute to our understanding of nitrogen fixation.

Presence and activity of nitrogen-fixing bacteria in Scots pine needles in a boreal forest: a nitrogen-addition experiment.

Endophytic nitrogen-fixing bacteria have been detected and isolated from the needles of conifer trees growing in North American boreal forests. Because boreal forests are nutrient-limited, these bacteria could provide an important source of nitrogen for tree species. This study aimed to determine their presence and activity in a Scandinavian boreal forest, using immunodetection of nitrogenase enzyme subunits and acetylene-reduction assays of native Scots pine (Pinus sylvestris L.) needles. The presence and rate of nitrogen fixation by endophytic bacteria were compared between control plots and fertilized plots in a nitrogen-addition experiment. In contrast to the expectation that nitrogen-fixation rates would decline in fertilized plots, as seen, for instance, with nitrogen-fixing bacteria associated with bryophytes, there was no difference in the presence or activity of nitrogen-fixing bacteria between the two treatments. The extrapolated calculated rate of nitrogen fixation relevant for the forest stand was 20gNha-1year-1, which is rather low compared with Scots pine annual nitrogen use but could be important for the nitrogen-poor forest in the long term. In addition, of 13 colonies of potential nitrogen-fixing bacteria isolated from the needles on nitrogen-free media, 10 showed in vitro nitrogen fixation. In summary, 16S rRNA sequencing identified the species as belonging to the genera Bacillus, Variovorax, Novosphingobium, Sphingomonas, Microbacterium and Priestia, which was confirmed by Illumina whole-genome sequencing. Our results confirm the presence of endophytic nitrogen-fixing bacteria in Scots pine needles and suggest that they could be important for the long-term nitrogen budget of the Scandinavian boreal forest.