Endophytic Bacterial Diversity in Rice (Oryza sativa L.) Roots Estimated by 16S rDNA Sequence Analysis

The endophytic bacterial diversity in the roots of rice (Oryza sativa L.) growing in the agricultural experimental station in Hebei Province, China was analyzed by 16S rDNA cloning, amplified ribosomal DNA restriction analysis (ARDRA), and sequence homology comparison. To effectively exclude the interference of chloroplast DNA and mitochondrial DNA of rice, a pair of bacterial PCR primers (799f–1492r) was selected to specifically amplify bacterial 16S rDNA sequences directly from rice root tissues. Among 192 positive clones in the 16S rDNA library of endophytes, 52 OTUs (Operational Taxonomic Units) were identified based on the similarity of the ARDRA banding profiles. Sequence analysis revealed diverse phyla of bacteria in the 16S rDNA library, which consisted of alpha, beta, gamma, delta, and epsilon subclasses of the Proteobacteria, Cytophaga/Flexibacter/Bacteroides (CFB) phylum, low G+C gram-positive bacteria, Deinococcus-Thermus, Acidobacteria, and archaea. The dominant group was Betaproteobacteria (27.08% of the total clones), and the most dominant genus was Stenotrophomonas. More than 14.58% of the total clones showed high similarity to uncultured bacteria, suggesting that nonculturable bacteria were detected in rice endophytic bacterial community. To our knowledge, this is the first report that archaea has been identified as endophytes associated with rice by the culture-independent approach. The results suggest that the diversity of endophytic bacteria is abundant in rice roots.

[1]  J. Kloepper,et al.  Interactions between Meloidogyne incognita and endophytic bacteria in cotton and cucumber , 1998 .

[2]  S. Sarrocco,et al.  Characterization of a free-living maize-rhizosphere population of Burkholderia cepacia: effect of seed treatment on disease suppression and growth promotion of maize , 1998 .

[3]  W. Song,et al.  Rice endophyte Pantoea agglomerans YS19 promotes host plant growth and affects allocations of host photosynthates , 2006, Journal of applied microbiology.

[4]  D. Lane.  16S/23S rRNA sequencing , 1991 .

[5]  R. Conrad,et al.  In Situ Stable Isotope Probing of Methanogenic Archaea in the Rice Rhizosphere , 2005, Science.

[6]  S. Tsuge,et al.  Evaluation of the Endophyte Enterobacter cloacae SM10 Isolated from Spinach Roots for Biological Control against Fusarium Wilt of Spinach , 2001, Journal of General Plant Pathology.

[7]  L. Forney,et al.  Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA , 1997, Applied and environmental microbiology.

[8]  M. Kimura.  A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences , 1980, Journal of Molecular Evolution.

[9]  James R. Cole,et al.  A new version of the RDP (Ribosomal Database Project) , 1999, Nucleic Acids Res..

[10]  A. Tripathi,et al.  Evaluation of plant growth promoting and colonization ability of endophytic diazotrophs from deep water rice. , 2001, Journal of biotechnology.

[11]  F. O'Gara,et al.  Biological control of Pythium ultimum by Stenotrophomonas maltophilia W81 is mediated by an extracellular proteolytic activity. , 1997, Microbiology.

[12]  W. Mahaffee,et al.  Bacterial endophytes in agricultural crops , 1997 .

[13]  S. Giovannoni,et al.  Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR , 1996, Applied and environmental microbiology.

[14]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[15]  M. Sakamoto,et al.  Movement and Fixation of Intestinal Microbiota after Administration of Human Feces to Germfree Mice , 2005, Applied and Environmental Microbiology.

[16]  G. Song,et al.  Preparation of DNA from Silica Gel Dried Mini-amount of Leaves of Oryza rufipogon for RAPD Study and Total DNA Bank Construction , 1999 .

[17]  J. Tissier,et al.  Physiological Characterization of Viable-but-Nonculturable Campylobacter jejuniCells , 1999, Applied and Environmental Microbiology.

[18]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[19]  A. Sessitsch,et al.  Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes. , 2002, FEMS microbiology ecology.

[20]  L. Cocolin,et al.  Direct Identification in Food Samples of Listeria spp. and Listeria monocytogenes by Molecular Methods , 2002, Applied and Environmental Microbiology.

[21]  K. Minamisawa,et al.  Endophytic Colonization and In Planta Nitrogen Fixation by a Herbaspirillum sp. Isolated from Wild Rice Species , 2001, Applied and Environmental Microbiology.

[22]  J. V. van Elsas,et al.  Diversity of Endophytic Bacterial Populations and Their Interaction with Xylella fastidiosa in Citrus Plants , 2002, Applied and Environmental Microbiology.

[23]  John Dunbar,et al.  Levels of Bacterial Community Diversity in Four Arid Soils Compared by Cultivation and 16S rRNA Gene Cloning , 1999, Applied and Environmental Microbiology.

[24]  M. Chelius,et al.  The Diversity of Archaea and Bacteria in Association with the Roots of Zea mays L. , 2001, Microbial Ecology.

[25]  E. Madsen,et al.  Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment , 1994, Applied and environmental microbiology.

[26]  W. Liesack,et al.  Novel Euryarchaeotal Lineages Detected on Rice Roots and in the Anoxic Bulk Soil of Flooded Rice Microcosms , 1998, Applied and Environmental Microbiology.

[27]  H. Blöcker,et al.  Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. , 1989, Nucleic acids research.

[28]  A. Pühler,et al.  Phylogenetic Analysis of Microbial Diversity in the Rhizoplane of Oilseed Rape (Brassica napus cv. Westar) Employing Cultivation-Dependent and Cultivation-Independent Approaches , 2001, Microbial Ecology.

[29]  J. Jansson,et al.  Modern Soil Microbiology , 2019 .

[30]  Xiuzhu Dong,et al.  Characterization of a novel plant growth-promoting bacteria strain Delftia tsuruhatensis HR4 both as a diazotroph and a potential biocontrol agent against various plant pathogens. , 2005, Systematic and applied microbiology.

[31]  J. Felsenstein.  CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP , 1985, Evolution; international journal of organic evolution.

[32]  W. Wenzel,et al.  Bacterial Communities Associated with Flowering Plants of the Ni Hyperaccumulator Thlaspi goesingense , 2004, Applied and Environmental Microbiology.

[33]  A. Chakrabarty,et al.  Biodegradation of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia strain AC1100: evolutionary insight. , 1996, Gene.

[34]  J. Vangronsveld,et al.  Endophytic Bacteria and Their Potential Applications , 2002 .

[35]  P. Oudemans,et al.  Characterization of a Chitinase Gene from Stenotrophomonas maltophilia Strain 34S1 and Its Involvement in Biological Control , 2002, Applied and Environmental Microbiology.

[36]  M. Cottrell,et al.  Community Composition of Marine Bacterioplankton Determined by 16S rRNA Gene Clone Libraries and Fluorescence In Situ Hybridization , 2000, Applied and Environmental Microbiology.

[37]  J. Baldani,et al.  Herbaspirillum frisingense sp. nov., a new nitrogen-fixing bacterial species that occurs in C4-fibre plants. , 2001, International journal of systematic and evolutionary microbiology.

[38]  R. Conrad,et al.  Detecting active methanogenic populations on rice roots using stable isotope probing. , 2005, Environmental microbiology.

[39]  P. Visca,et al.  Characterization of two rhizosphere isolates of Pseudomonas cepacia and their plant growth promoting activity , 1993 .

[40]  E. Stackebrandt,et al.  Nucleic acid techniques in bacterial systematics , 1991 .

[41]  H. Heuer,et al.  Application of denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis for studying soil microbial communities. , 1997 .

[42]  I. Good.  THE POPULATION FREQUENCIES OF SPECIES AND THE ESTIMATION OF POPULATION PARAMETERS , 1953 .

[43]  Hans H. Cheng,et al.  Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA , 1997, Applied and environmental microbiology.

[44]  E. Stackebrandt,et al.  Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species , 1995, Applied and environmental microbiology.

[45]  K. Minamisawa,et al.  Anaerobic Nitrogen-Fixing Consortia Consisting of Clostridia Isolated from Gramineous Plants , 2004, Applied and Environmental Microbiology.

[46]  H. Heuer,et al.  Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed , 2001, Applied and Environmental Microbiology.

[47]  J. Kloepper,et al.  Survey of indigenous bacterial endophytes from cotton and sweet corn , 1995, Plant and Soil.

[48]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.