Nitrogen fixation and phytohormone stimulation of sugarcane plant through plant growth promoting diazotrophic Pseudomonas.

Diazotrophic microorganisms are free-living groups of organisms that can convert atmospheric nitrogen (N) into bioavailable nitrogen for plants, which increases crop development and production. The purpose of the current study was to ascertain how diazotrophic plant growth promoting (PGP) Pseudomonas strains (P. koreensis CY4 and P. entomophila CN11) enhanced nitrogen fixation, defense activity, and PGP attributes of sugarcane varieties; GT11 and G×B9. A 15N isotope-dilution study was conducted to confirm the sugarcane strains' capacity to fix nitrogen, and the results indicated that between 21 to 35% of plant, nitrogen is fixed biologically by selected rhizobacteria. In comparison to the control, after 30, 60, and 90 days, both CY4 and CN11 strains significantly increased defense-related enzymes (catalase, peroxidase, phenylalanine ammonia-lyase, superoxide dismutase, glucanase, and chitinase) and phytohormones (abscisic acid, ABA, cytokinin, etc.) in GT11 and GXB. Additionally, the expression of SuCHI, SuGLU, SuCAT, SuSOD, and SuPAL genes was found to be elevated in Pseudomonas strains inoculated plants using real-time quantitative polymerase chain reaction (RT-qPCR). Both bacterial strains increased all physiological parameters and chlorophyll content in sugarcane plants more than their control. The effects of P. koreensis CY4 and P. entomophila CN11 strains on sugarcane growth promotion and nitrogen fixation under greenhouse conditions are described here for the first time systematically. The results of confirmation studies demonstrated that P. koreensis CY4 and P. entomophila are PGP bacterial strains with the potential to be employed as a biofertilizer for sugarcane growth, nitrogen nutrient absorption, and reduced application of chemical nitrogenous fertilizers in agricultural fields. .

Diversity of nitrogen-fixing rhizobacteria associated with sugarcane: a comprehensive study of plant-microbe interactions for growth enhancement in Saccharum spp.

BACKGROUND: Nitrogen 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. RESULTS: In 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. CONCLUSION: The 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.

Diazotrophic Bacteria Pantoea dispersa and Enterobacter asburiae Promote Sugarcane Growth by Inducing Nitrogen Uptake and Defense-Related Gene Expression.

Sugarcane is a major crop in tropical and subtropical regions of the world. In China, the application of large amounts of nitrogen (N) fertilizer to boost sugarcane yield is commonplace, but it causes substantial environmental damages, particularly soil, and water pollution. Certain rhizosphere microbes are known to be beneficial for sugarcane production, but much of the sugarcane rhizosphere microflora remains unknown. We have isolated several sugarcane rhizosphere bacteria, and 27 of them were examined for N-fixation, plant growth promotion, and antifungal activity. 16S rRNA gene sequencing was used to identify these strains. Among the isolates, several strains were found to have a relatively high activity of nitrogenase and ACC deaminase, the enzyme that reduces ethylene production in plants. These strains were found to possess nifH and acdS genes associated with N-fixation and ethylene production, respectively. Two of these strains, Pantoea dispersa-AA7 and Enterobacter asburiae-BY4 showed maximum plant growth promotion (PGP) and nitrogenase activity, and thus they were selected for detailed analysis. The results show that they colonize different sugarcane tissues, use various growth substrates (carbon and nitrogen), and tolerate various stress conditions (pH and osmotic stress). The positive effect of AA7 and BY4 strains on nifH and stress-related gene (SuCAT, SuSOD, SuPAL, SuCHI, and SuGLU) expression and the induction of defense-related processes in two sugarcane varieties, GT11 and GXB9, showed their potential for stress amelioration and PGP. Both bacterial strains increased several sugarcane physiological parameters. i.e., plant height, shoot weight, root weight, leaf area, chlorophyll content, and photosynthesis, in plants grown under greenhouse conditions. The ability of rhizobacteria on N-fixing in sugarcane was also confirmed by a 15N isotope-dilution study, and the estimate indicates a contribution of 21-35% of plant nitrogen by rhizobacterial biological N fixation (BNF). This is the first report of sugarcane growth promotion by N-fixing rhizobacteria P. dispersa and E. asburiae strains. Both strains could be used as biofertilizer for sugarcane to minimize nitrogen fertilizer use and better disease management.

Methods for Estimation of Nitrogen Components in Plants and Microorganisms.

Nitrogen (N2) is the most necessary element in the atmosphere, it is an energetic micronutrient for plant growth and development after water, besides its key role in chlorophyll production, which is crucial for photosynthesis process. Biological nitrogen fixation is measured to be the most potent method to deliver a fixed way of nitrogen to the plants. Plant depends on free-living and symbiotic microbes present in the soil for nitrogen because it cannot be absorbed by the plant itself directly from the atmosphere. Many techniques were reported in the laboratory for nitrogen estimation till now, but Kjeldahl digestion and acetylene reduction assay (ARA) techniques became the most popular. In this chapter, we focus on the most common and popular methods used to determine plant N2; awareness obtained through the wide application of these methods should offer the source for the N2 fixation rate in agriculture system.

Proteomic Analysis of the Resistance Mechanisms in Sugarcane during Sporisorium scitamineum Infection.

Smut disease is caused by Sporisorium scitamineum, an important sugarcane fungal pathogen causing an extensive loss in yield and sugar quality. The available literature suggests that there are two types of smut resistance mechanisms: external resistance by physical or chemical barriers and intrinsic internal resistance mechanisms operating at host⁻pathogen interaction at cellular and molecular levels. The nature of smut resistance mechanisms, however, remains largely unknown. The present study investigated the changes in proteome occurring in two sugarcane varieties with contrasting susceptibility to smut-F134 and NCo310-at whip development stage after S. scitamineum infection. Total proteins from pathogen inoculated and uninoculated (control) leaves were separated by two-dimensional gel electrophoresis (2D-PAGE). Protein identification was performed using BLASTp and tBLASTn against NCBI nonredundant protein databases and EST databases, respectively. A total of thirty proteins spots representing differentially expressed proteins (DEPs), 16 from F134 and 14 from NCo310, were identified and analyzed by MALDI-TOF/TOF MS. In F134, 4 DEPs were upregulated and nine were downregulated, while, nine were upregulated and three were downregulated in NCo310. The DEPs were associated with DNA binding, metabolic processes, defense, stress response, photorespiration, protein refolding, chloroplast, nucleus and plasma membrane. Finally, the expression of CAT, SOD, and PAL with recognized roles in S. scitamineum infection in both sugarcane verities were analyzed by real-time quantitative PCR (RT-qPCR) technique. Identification of genes critical for smut resistance in sugarcane will increase our knowledge of S. scitamineum-sugarcane interaction and help to develop molecular and conventional breeding strategies for variety improvement.

Genetic Diversity of Nitrogen-Fixing and Plant Growth Promoting Pseudomonas Species Isolated from Sugarcane Rhizosphere.

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.