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Unraveling Nitrogen Fixing Potential of Endophytic Diazotrophs of Different Saccharum Species for Sustainable Sugarcane Growth.

ABSTRACT: Sugarcane (Saccharum officinarum L.) is one of the world's highly significant commercial crops. The amounts of synthetic nitrogen (N₂) fertilizer required to grow the sugarcane plant at its initial growth stages are higher, which increases the production costs and adverse environmental consequences globally. To combat this issue, sustainable environmental and economic concerns among researchers are necessary. The endophytic diazotrophs can offer significant amounts of nitrogen to crops through the biological nitrogen fixation mediated nif gene. The nifH gene is the most extensively utilized molecular marker in nature for studying N₂ fixing microbiomes. The present research intended to determine the existence of novel endophytic diazotrophs through culturable and unculturable bacterial communities (EDBCs). The EDBCs of different tissues (root, stem, and leaf) of five sugarcane cultivars (Saccharum officinarum L. cv. Badila, S. barberi Jesw.cv Pansahi, S. robustum, S. spontaneum, and S. sinense Roxb.cv Uba) were isolated and molecularly characterized to evaluate N₂ fixation ability. The diversity of EDBCs was observed based on nifH gene Illumina MiSeq sequencing and a culturable approach. In this study, 319766 operational taxonomic units (OTUs) were identified from 15 samples. The minimum number of OTUs was recorded in leaf tissues of S. robustum and maximum reads in root tissues of S. spontaneum. These data were assessed to ascertain the structure, diversity, abundance, and relationship between the microbial community. A total of 40 bacterial families with 58 genera were detected in different sugarcane species. Bacterial communities exhibited substantially different alpha and beta diversity. In total, 16 out of 20 genera showed potent N₂-fixation in sugarcane and other crops. According to principal component analysis (PCA) and hierarchical clustering (Bray-Curtis dis) evaluation of OTUs, bacterial microbiomes associated with root tissues differed significantly from stem and leaf tissues of sugarcane. Significant differences often were observed in EDBCs among the sugarcane tissues. We tracked and validated the plethora of individual phylum strains and assessed their nitrogenase activity with a culture-dependent technique. The current work illustrated the significant and novel results of many uncharted endophytic microbial communities in different tissues of sugarcane species, which provides an experimental system to evaluate the biological nitrogen fixation (BNF) mechanism in sugarcane. The novel endophytic microbial communities with N₂-fixation ability play a remarkable and promising role in sustainable agriculture production.

SUBMITTER: Singh RK

PROVIDER: S-EPMC9181200 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Unraveling Nitrogen Fixing Potential of Endophytic Diazotrophs of Different Saccharum Species for Sustainable Sugarcane Growth.

Singh Rajesh Kumar RK Singh Pratiksha P Sharma Anjney A Guo Dao-Jun DJ Upadhyay Sudhir K SK Song Qi-Qi QQ Verma Krishan K KK Li Dong-Ping DP Malviya Mukesh Kumar MK Song Xiu-Peng XP Yang Li-Tao LT Li Yang-Rui YR

International journal of molecular sciences 20220602 11

Sugarcane (Saccharum officinarum L.) is one of the world's highly significant commercial crops. The amounts of synthetic nitrogen (N2) fertilizer required to grow the sugarcane plant at its initial growth stages are higher, which increases the production costs and adverse environmental consequences globally. To combat this issue, sustainable environmental and economic concerns among researchers are necessary. The endophytic diazotrophs can offer significant amounts of nitrogen ...[more]

PMID: 35682919

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Project description:Endophytic nitrogen-fixing bacteria are versatile and widely distributed in plants. Numerous strains of endophytic nitrogen-fixing bacteria are used as biofertilizers to minimize the utilization of chemical fertilizers, improve nutrient use efficiency, increase crop productivity, and reduce environmental pollution. However, the mechanism underlying the interaction between nitrogen-fixing bacteria and plants is still unclear. So, the present study was planned to assess the effects of endophytic nitrogen-fixing bacteria on sugarcane by analyzing the changes in physiological and biochemical activities. In the current study, Klebsiella variicola DX120E, an endophytic nitrogen-fixing bacterium, was inoculated on sugarcane varieties B8 and ROC22 to evaluate the effects on nitrogen and carbon metabolism-related enzymatic activity and biomass. Results showed that DX120E inoculation improved the enzymatic activities related to gluconeogenesis and nitrogen metabolism increased the sugarcane plant's height, cane juice Brix, biomass, chlorophyll, and soluble sugar content in sugarcane. Metabolomics analysis revealed that the metabolome modules were highly enriched in carbon and nitrogen metabolic pathways of strain-affected sugarcane than uninoculated control. The identified carbohydrates were associated with the glycolysis or gluconeogenesis and tricarboxylic acid (TCA) cycle in plants. Metabolomic profiling in the present investigation showed that carbohydrate metabolism is coordinated with nitrogen metabolism to provide carbon skeletons and energy to amino acid synthesis, and amino acid degradation results in several metabolites used by the citric acid cycle as an energy source. Moreover, differentially expressed metabolites of non-proteinogenic amino acids have a further complementary role to the action of endophytic nitrogen-fixing bacteria. Meanwhile, a significant difference in metabolites and metabolic pathways present in stems and leaves of B8 and ROC22 varieties was found. This study discovered the potential benefits of DX120E in sugarcane and suggested candidate regulatory elements to enhance interactions between nitrogen-fixing microbes and sugarcane.

Project description:BackgroundNitrogen is an essential element for sugarcane growth and development and is generally applied in the form of urea often much more than at recommended rates, causing serious soil degradation, particularly soil acidification, as well as groundwater and air pollution. In spite of the importance of nitrogen for plant growth, fewer reports are available to understand the application and biological role of N2 fixing bacteria to improve N2 nutrition in the sugarcane plant.ResultsIn this study, a total of 350 different bacterial strains were isolated from rhizospheric soil samples of the sugarcane plants. Out of these, 22 isolates were selected based on plant growth promotion traits, biocontrol, and nitrogenase activity. The presence and activity of the nifH gene and the ability of nitrogen-fixation proved that all 22 selected strains have the ability to fix nitrogen. These strains were used to perform 16S rRNA and rpoB genes for their identification. The resulted amplicons were sequenced and phylogenetic analysis was constructed. Among the screened strains for nitrogen fixation, CY5 (Bacillus megaterium) and CA1 (Bacillus mycoides) were the most prominent. These two strains were examined for functional diversity using Biolog phenotyping, which confirmed the consumption of diverse carbon and nitrogen sources and tolerance to low pH and osmotic stress. The inoculated bacterial strains colonized the sugarcane rhizosphere successfully and were mostly located in root and leaf. The expression of the nifH gene in both sugarcane varieties (GT11 and GXB9) inoculated with CY5 and CA1 was confirmed. The gene expression studies showed enhanced expression of genes of various enzymes such as catalase, phenylalanine-ammonia-lyase, superoxide dismutase, chitinase and glucanase in bacterial-inoculated sugarcane plants.ConclusionThe results showed that a substantial number of Bacillus isolates have N-fixation and biocontrol property against two sugarcane pathogens Sporisorium scitamineum and Ceratocystis paradoxa. The increased activity of genes controlling free radical metabolism may at least in part accounts for the increased tolerance to pathogens. Nitrogen-fixation was confirmed in sugarcane inoculated with B. megaterium and B. mycoides strains using N-balance and 15N2 isotope dilution in different plant parts of sugarcane. This is the first report of Bacillus mycoides as a nitrogen-fixing rhizobacterium in sugarcane.

Project description:The study was designed to isolate and characterize Pseudomonas spp. from sugarcane rhizosphere, and to evaluate their plant- growth- promoting (PGP) traits and nitrogenase activity. A biological nitrogen-fixing microbe has great potential to replace chemical fertilizers and be used as a targeted biofertilizer in a plant. A total of 100 isolates from sugarcane rhizosphere, belonging to different species, were isolated; from these, 30 isolates were selected on the basis of preliminary screening, for in vitro antagonistic activities against sugarcane pathogens and for various PGP traits, as well as nitrogenase activity. The production of IAA varied from 312.07 to 13.12 μg mL-1 in tryptophan supplemented medium, with higher production in AN15 and lower in CN20 strain. The estimation of ACC deaminase activity, strains CY4 and BA2 produced maximum and minimum activity of 77.0 and 15.13 μmoL mg-1 h-1. For nitrogenase activity among the studied strains, CoA6 fixed higher and AY1 fixed lower in amounts (108.30 and 6.16 μmoL C2H2 h-1 mL-1). All the strains were identified on the basis of 16S rRNA gene sequencing, and the phylogenetic diversity of the strains was analyzed. The results identified all strains as being similar to Pseudomonas spp. Polymerase chain reaction (PCR) amplification of nifH and antibiotic genes was suggestive that the amplified strains had the capability to fix nitrogen and possessed biocontrol activities. Genotypic comparisons of the strains were determined by BOX, ERIC, and REP PCR profile analysis. Out of all the screened isolates, CY4 (Pseudomonas koreensis) and CN11 (Pseudomonas entomophila) showed the most prominent PGP traits, as well as nitrogenase activity. Therefore, only these two strains were selected for further studies; Biolog profiling; colonization through green fluorescent protein (GFP)-tagged bacteria; and nifH gene expression using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The Biolog phenotypic profiling, which comprised utilization of C and N sources, and tolerance to osmolytes and pH, revealed the metabolic versatility of the selected strains. The colonization ability of the selected strains was evaluated by genetically tagging them with a constitutively expressing GFP-pPROBE-pTetr-OT plasmid. qRT-PCR results showed that both strains had the ability to express the nifH gene at 90 and 120 days, as compared to a control, in both sugarcane varieties GT11 and GXB9. Therefore, our isolated strains, P. koreensis and P. entomophila may be used as inoculums or in biofertilizer production for enhancing growth and nutrients, as well as for improving nitrogen levels, in sugarcane and other crops. The present study, to the best of our knowledge, is the first report on the diversity of Pseudomonas spp. associated with sugarcane in Guangxi, China.

Project description:Sugarcane is an important sugar and energy crop worldwide, requiring a large amount of nitrogen (N). However, excessive application of synthetic N fertilizer causes environmental pollution in farmland. Endophytic nitrogen-fixing bacteria (ENFB) provide N nutrition for plants through biological N fixation, thus reducing the need for chemical fertilizers. The present study investigated the effect of the N-fixing endophytic strain Enterobacter roggenkampii ED5 on phytohormone indole-3-acetic acid (IAA), N-metabolism enzyme activities, microbial community compositions, and N cycle genes in sugarcane rhizosphere soil at different N levels. Three levels of 15N-urea, such as low N (0 kg/ha), medium N (150 kg/ha), and high N (300 kg/ha), were applied. The results showed that, after inoculating strain ED5, the IAA content in sugarcane leaves was significantly increased by 68.82% under low N condition at the seedling stage (60 days). The nitrate reductase (NR) activity showed a downward trend. However, the glutamine synthase (GS) and NADH-glutamate dehydrogenase (NADH-GDH) activities were significantly enhanced compared to the control under the high N condition, and the GS and NR genes had the highest expression at 180 and 120 days, respectively, at the low N level. The total N content in the roots, stems, and leaves of sugarcane was higher than the control. The 15N atom % excess of sugarcane decreased significantly under medium N condition, indicating that the medium N level was conducive to N fixation in strain ED5. Metagenome analysis of sugarcane rhizosphere soil exhibited that the abundance of N-metabolizing microbial richness was increased under low and high N conditions after inoculation of strain ED5 at the genus level, while it was increased at the phylum level only under the low N condition. The LefSe (LDA > 2, p < 0.05) found that the N-metabolism-related differential microorganisms under the high N condition were higher than those under medium and low N conditions. It was also shown that the abundance of nifDHK genes was significantly increased after inoculation of ED5 at the medium N level, and other N cycle genes had high abundance at the high N level after inoculation of strain ED5. The results of this study provided a scientific reference for N fertilization in actual sugarcane production.

Dataset Information

Unraveling Nitrogen Fixing Potential of Endophytic Diazotrophs of Different Saccharum Species for Sustainable Sugarcane Growth.

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Unraveling Nitrogen Fixing Potential of Endophytic Diazotrophs of Different <i>Saccharum</i> Species for Sustainable Sugarcane Growth.

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