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      Plant Growth-Promoting Rhizobacteria Inoculation to Enhance Vegetative Growth, Nitrogen Fixation and Nitrogen Remobilisation of Maize under Greenhouse Conditions

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          Abstract

          Plant growth-promoting rhizobacteria (PGPR) may provide a biological alternative to fix atmospheric N 2 and delay N remobilisation in maize plant to increase crop yield, based on an understanding that plant-N remobilisation is directly correlated to its plant senescence. Thus, four PGPR strains were selected from a series of bacterial strains isolated from maize roots at two locations in Malaysia. The PGPR strains were screened in vitro for their biochemical plant growth-promoting (PGP) abilities and plant growth promotion assays. These strains were identified as Klebsiella sp. Br1, Klebsiella pneumoniae Fr1, Bacillus pumilus S1r1 and Acinetobacter sp. S3r2 and a reference strain used was Bacillus subtilis UPMB10. All the PGPR strains were tested positive for N 2 fixation, phosphate solubilisation and auxin production by in vitro tests. In a greenhouse experiment with reduced fertiliser-N input (a third of recommended fertiliser-N rate), the N 2 fixation abilities of PGPR in association with maize were determined by 15N isotope dilution technique at two harvests, namely, prior to anthesis (D 50) and ear harvest (D 65). The results indicated that dry biomass of top, root and ear, total N content and bacterial colonisations in non-rhizosphere, rhizosphere and endosphere of maize roots were influenced by PGPR inoculation. In particular, the plants inoculated with B. pumilus S1r1 generally outperformed those with the other treatments. They produced the highest N 2 fixing capacity of 30.5% (262 mg N 2 fixed plant −1) and 25.5% (304 mg N 2 fixed plant −1) of the total N requirement of maize top at D 50 and D 65, respectively. N remobilisation and plant senescence in maize were delayed by PGPR inoculation, which is an indicative of greater grain production. This is indicated by significant interactions between PGPR strains and time of harvests for parameters on N uptake and at. % 15N e of tassel. The phenomenon is also supported by the lower N content in tassels of maize treated with PGPR, namely, B. pumilus S1r1, K. pneumoniae Fr1, B. subtilis UPMB10 and Acinetobacter sp. S3r2 at D 65 harvest. This study provides evidence that PGPR inoculation, namely, B. pumilus S1r1 can biologically fix atmospheric N 2 and provide an alternative technique, besides plant breeding, to delay N remobilisation in maize plant for higher ear yield (up to 30.9%) with reduced fertiliser-N input.

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          Most cited references13

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          16S ribosomal DNA amplification for phylogenetic study.

          A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.
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            Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture.

            Productive agriculture needs a large amount of expensive nitrogenous fertilizers. Improving nitrogen use efficiency (NUE) of crop plants is thus of key importance. NUE definitions differ depending on whether plants are cultivated to produce biomass or grain yields. However, for most plant species, NUE mainly depends on how plants extract inorganic nitrogen from the soil, assimilate nitrate and ammonium, and recycle organic nitrogen. Efforts have been made to study the genetic basis as well as the biochemical and enzymatic mechanisms involved in nitrogen uptake, assimilation, and remobilization in crops and model plants. The detection of the limiting factors that could be manipulated to increase NUE is the major goal of such research. An overall examination of the physiological, metabolic, and genetic aspects of nitrogen uptake, assimilation and remobilization is presented in this review. The enzymes and regulatory processes manipulated to improve NUE components are presented. Results obtained from natural variation and quantitative trait loci studies are also discussed. This review presents the complexity of NUE and supports the idea that the integration of the numerous data coming from transcriptome studies, functional genomics, quantitative genetics, ecophysiology and soil science into explanatory models of whole-plant behaviour will be promising.
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              The Diversity of Archaea and Bacteria in Association with the Roots of Zea mays L.

              The diversity of bacteria and archaea associating on the surface and interior of maize roots (Zea mays L.) was investigated. A bacterial 16S rDNA primer was designed to amplify bacterial sequences directly from maize roots by PCR to the exclusion of eukaryotic and chloroplast DNA. The mitochondrial sequence from maize was easily separated from the PCR-amplified bacterial sequences by size fractionation. The culturable component of the bacterial community was also assessed, reflecting a community composition different from that of the clone library. The phylogenetic overlap between organisms obtained by cultivation and those identified by direct PCR amplification of 16S rDNA was 48%. Only 4 bacterial divisions were found in the culture collection, which represented 27 phylotypes, whereas 6 divisions were identified in the clonal analysis, comprising 74 phylotypes, including a member of the OP10 candidate division originally described as a novel division level lineage in a Yellowstone hot spring. The predominant group in the culture collection was the actinobacteria and within the clone library, the a-proteobacteria predominated. The population of maize-associated proteobacteria resembled the proteobacterial population of a typical soil community within which resided a subset of specific plant-associated bacteria, such as Rhizobium- and Herbaspirillum-related phylotypes. The representation of phylotypes within other divisions (OP10 and Acidobacterium) suggests that maize roots support a distinct bacterial community. The diversity within the archaeal domain was low. Of the 50 clones screened, 6 unique sequence types were identified, and 5 of these were highly related to each other (sharing 98% sequence identity). The archaeal sequences clustered with good bootstrap support near Marine group I (crenarchaea) and with Marine group II (euryarchaea) uncultured archaea. The results suggest that maize supports a diverse root-associated microbial community composed of species that for the first time have been described as inhabitants of a plant-root environment.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                24 March 2016
                2016
                : 11
                : 3
                : e0152478
                Affiliations
                [1 ]Department of Land Management, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
                [2 ]Agrotechnology and Biosciences Division, Malaysian Nuclear Agency, Bangi, Selangor, Malaysia
                Estación Experimental del Zaidín (CSIC), SPAIN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: KBK RO KAR ZHS. Performed the experiments: KBK ZHS. Analyzed the data: KBK KAR ZHS. Contributed reagents/materials/analysis tools: KBK RO KAR ZHS. Wrote the paper: KBK RO KAR ZHS.

                Article
                PONE-D-15-45829
                10.1371/journal.pone.0152478
                4807084
                27011317
                91b42add-057e-4870-808b-bc859f313458
                © 2016 Kuan et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 19 October 2015
                : 15 March 2016
                Page count
                Figures: 1, Tables: 7, Pages: 19
                Funding
                K.B. Kuan was supported by the MyBrain15 and Graduate Research Fellowship scholarships from the Ministry of Education Malaysia and Universiti Putra Malaysia, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Agriculture
                Crop Science
                Crops
                Cereal Crops
                Maize
                Biology and Life Sciences
                Organisms
                Plants
                Grasses
                Maize
                Research and Analysis Methods
                Model Organisms
                Plant and Algal Models
                Maize
                Biology and Life Sciences
                Plant Science
                Plant Anatomy
                Leaves
                Biology and Life Sciences
                Organisms
                Bacteria
                Klebsiella
                Klebsiella Pneumoniae
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Klebsiella
                Klebsiella Pneumoniae
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Klebsiella
                Klebsiella Pneumoniae
                Biology and Life Sciences
                Organisms
                Bacteria
                Bacillus
                Bacillus Subtilis
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Bacillus
                Bacillus Subtilis
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Bacillus
                Bacillus Subtilis
                Research and Analysis Methods
                Model Organisms
                Prokaryotic Models
                Bacillus Subtilis
                Biology and Life Sciences
                Organisms
                Bacteria
                Acinetobacter
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Acinetobacter
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Acinetobacter
                Research and Analysis Methods
                Database and Informatics Methods
                Biological Databases
                Sequence Databases
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Databases
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Databases
                Biology and Life Sciences
                Developmental Biology
                Plant Growth and Development
                Biology and Life Sciences
                Plant Science
                Plant Growth and Development
                Physical Sciences
                Chemistry
                Chemical Compounds
                Phosphates
                Custom metadata
                All relevant data are within the paper and its supporting information files. All nucleotide sequence files are available from the NCBI database (accession number KP257586, KP295962, KP295963, KP641617, KP641618).

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