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Plant growth-promoting nitrogen-fixing enterobacteria are in association with sugarcane plants growing in Guangxi, China.

ABSTRACT: The current nitrogen fertilization for sugarcane production in Guangxi, the major sugarcane-producing area in China, is very high. We aim to reduce nitrogen fertilization and improve sugarcane production in Guangxi with the help of indigenous sugarcane-associated nitrogen-fixing bacteria. We initially obtained 196 fast-growing bacterial isolates associated with the main sugarcane cultivar ROC22 plants in fields using a nitrogen-deficient minimal medium and screened out 43 nitrogen-fixing isolates. Analysis of 16S rRNA gene sequences revealed that 42 of the 43 nitrogen-fixing isolates were affiliated with the genera Enterobacter and Klebsiella. Most of the nitrogen-fixing enterobacteria possessed two other plant growth-promoting activities of IAA production, siderophore production and phosphate solubilization. Two Enterobacter spp. strains of NN145S and NN143E isolated from rhizosphere soil and surface-sterilized roots, respectively, of the same ROC22 plant were used to inoculate micropropagated sugarcane plantlets. Both strains increased the biomass and nitrogen content of the sugarcane seedlings grown with nitrogen fertilization equivalent to 180 kg urea ha(-1), the recommended nitrogen fertilization for ROC22 cane crops at the seedling stage. (15)N isotope dilution assays demonstrated that biological nitrogen fixation contributed to plant growth promotion. These results suggested that indigenous nitrogen-fixing enterobacteria have the potential to fix N(2) associated with sugarcane plants grown in fields in Guangxi and to improve sugarcane production.

SUBMITTER: Lin L

PROVIDER: S-EPMC4103546 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Plant growth-promoting nitrogen-fixing enterobacteria are in association with sugarcane plants growing in Guangxi, China.

Lin Li L Li Zhengyi Z Hu Chunjin C Zhang Xincheng X Chang Siping S Yang Litao L Li Yangrui Y An Qianli Q

Microbes and environments 20120418 4

The current nitrogen fertilization for sugarcane production in Guangxi, the major sugarcane-producing area in China, is very high. We aim to reduce nitrogen fertilization and improve sugarcane production in Guangxi with the help of indigenous sugarcane-associated nitrogen-fixing bacteria. We initially obtained 196 fast-growing bacterial isolates associated with the main sugarcane cultivar ROC22 plants in fields using a nitrogen-deficient minimal medium and screened out 43 nitrogen-fixing isolate ...[more]

PMID: 22510648

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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 the leading economic crop in China, requires huge quantities of nitrogen in the preliminary plant growth stages. However, the use of an enormous amount of nitrogen fertilizer increases the production price, and have detrimental results on the environment, causes severe soil and water pollution. In this study, a total of 175 endophytic strains were obtained from the sugarcane roots, belonging to five different species, i.e., Saccharum officinarum, Saccharum barberi, Saccharum robustum, Saccharum spontaneum, and Saccharum sinense. Among these, only 23 Enterobacter strains were chosen based on nitrogen fixation, PGP traits, hydrolytic enzymes production, and antifungal activities. Also, all selected strains were showed diverse growth range under different stress conditions, i.e., pH (5-10), temperature (20-45°C), and NaCl (7-12%) and 14 strains confirmed positive nifH, and 12 strains for acdS gene amplification, suggested that these strains could fix nitrogen along with stress tolerance properties. Out of 23 selected strains, Enterobacter roggenkampii ED5 was the most potent strain. Hence, this strain was further selected for comprehensive genome analysis, which includes a genome size of 4,702,851 bp and 56.05% of the average G + C content. Genome annotations estimated 4349 protein-coding with 83 tRNA and 25 rRNA genes. The CDSs number allocated to the KEGG, COG, and GO database were 2839, 4028, and 2949. We recognized a total set of genes that are possibly concerned with ACC deaminase activity, siderophores and plant hormones production, nitrogen and phosphate metabolism, symbiosis, root colonization, biofilm formation, sulfur assimilation and metabolism, along with resistance response toward a range of biotic and abiotic stresses. E. roggenkampii ED5 strain was also a proficient colonizer in sugarcane (variety GT11) and enhanced growth of sugarcane under the greenhouse. To the best of our knowledge, this is the first information on the whole-genome sequence study of endophytic E. roggenkampii ED5 bacterium associated with sugarcane root. And, our findings proposed that identification of predicted genes and metabolic pathways might describe this strain an eco-friendly bioresource to promote sugarcane growth by several mechanisms of actions under multi-stresses.

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:Root endophytic nitrogen-fixing bacteria (reNFB) have been proposed as important contributors to the invasiveness of exotic legumes; however, the reNFB of invasive nonlegumes has received less attention. In particular, the growth-promoting effect of reNFB on invasive plants remains unknown. In this study, 131 strains of potential nitrogen-fixing bacteria were isolated and purified from the roots of the invasive plant, Ageratina adenophora, in Southwest China. Phylogenetically, these reNFB were categorized into three phyla at 97% sequence identity that included Proteobacteria (92.4%), Actinobacteria (4.6%), and Firmicutes (3.1%). The dominant isolates ranked by number were Pseudomonas (80 isolates, 61.1%), Rhizobium (12 isolates, 9.2%), and Duganella (11 isolates, 8.4%). The community composition and diversity of A. adenophora reNFB were markedly different across study regions. The capacity of these reNFB to accumulate indolyl-3-acetic acid (IAA), solubilize phosphate, and produce siderophores was determined. All 131 isolates of reNFB accumulated IAA, 67 isolates solubilized phosphate, and 108 isolates produced siderophores. Among the three dominant genera of reNFB, Pseudomonas had the highest phosphorus solubilization and siderophore production, while the accumulation of IAA in the genus Duganella was the lowest. Interestingly, the calculated reNFB Shannon diversity index of each A. adenophora individual was negatively correlated with the capacity of reNFB to produce growth-promoting products. Six randomly selected isolates from three dominant genera were further used to conduct inoculation experiments, and all isolates showed significant positive growth-promoting effects on A. adenophora seedlings. The contribution of reNFB to the root biomass was higher than that to the shoot biomass. Our results suggest that reNFB, similar to soil or nodular nitrogen-fixing bacteria, can potentially promote plant growth and may play an important role in the invasion of nonleguminous plants. More detailed studies on the correlation between reNFB and invasive plants are necessary.

Dataset Information

Plant growth-promoting nitrogen-fixing enterobacteria are in association with sugarcane plants growing in Guangxi, China.

Publications

Plant growth-promoting nitrogen-fixing enterobacteria are in association with sugarcane plants growing in Guangxi, China.

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