Genetic diversity of N2-fixing bacteria associated with rice roots by molecular evolutionary analysis of a nifD library

The rhizosphere of wetland rice has significant N2-fixing activity. It has been suggested that N2 fixation in the rice root zone is associated with the activity of various N2-fixing heterotrophic bacteria that inhabit the rice rhizosphere. Because of the generic diversity, many different isolation media and conditions are required to count and isolate these bacteria. In an attempt to overcome any bias from culture-dependent methods we amplified nifD segments from crude rice root DNA by the polymerase chain reaction. The nifD fragments were then cloned into a pT7 BlueT-vector to construct a nifD library. Sixteen cloned nifD genes chosen at random from the library were sequenced. A comparison with published sequences indicated the presence of seven novel groups of NifD proteins, which implies the existence of at least seven components in the diazotrophic community of rice roots, dominated mainly by proteobacteria. We also observed genetic variability within the clusters, which suggests the coexistence of many closely related bacterial lineages. However, we did not find Azospirillum-like nifD clones, although many reports indicated the widespread presence of Azospirillum spp. Therefore, it remains to be clarified whether Azospirillum species are the widespread N2-fixing bacteria in rice roots.Key words: N2 fixation, molecular evolution, nifD, rice rhizosphere.

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Genetic diversity analysis of diazotrophs in the rice rhizosphere

Chinese Journal of Agricultural Biotechnology ◽

10.1017/s1479236207001982 ◽

2007 ◽

Vol 4 (3) ◽

pp. 253-258 ◽

Cited By ~ 3

Author(s):

Chen Bin ◽

Zheng Si-Ping ◽

Zhou Li-Juan ◽

Lin Zhi-Min ◽

Song Ya-Na ◽

...

Keyword(s):

Genetic Diversity ◽

Fragment Length Polymorphism ◽

Rhizospheric Soil ◽

Chain Reaction ◽

Rice Roots ◽

Rice Rhizosphere ◽

Pcr Rflp ◽

Pcr Products ◽

Polymerase Chain ◽

Diazotrophic Community

SUMMARYThe genetic diversity of dinitrogen-fixing bacteria associated with rice (Oryza sativa) was assessed by a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) approach on thenifHgene amplified directly from DNA extracted from washed rice roots and rhizospheric soil. Restriction digestion with the enzymesMnlI andHaeIII was performed to characterize 54 clonednifHPCR products. RFLP profiles were clustered and analysed with the UPGMA program. Eight pairs of similar RFLP patterns (similarity>50%) and two pairs of homologous RFLP patterns (100% identity) were found from the washed roots and the rhizospheric soil, respectively. Three specific diazotrophic patterns were found from rhizospheric soil and rice roots. The analyses have revealed the presence of differentnifHtypes, which appear to be significant components of the diazotrophic community in paddy fields, indicating that some of the diazotrophs may colonize the inside and the surface of the rice roots.

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Effectiveness of complex inoculation of spring wheat with N[2] -fi xing bacteria Azospirillum brasilense and mold-antagonist Chaetomium cochliodes

Agricultural science and practice ◽

10.15407/agrisp1.03.057 ◽

2014 ◽

Vol 1 (3) ◽

pp. 57-61

Author(s):

E. Kopylov

Keyword(s):

Spring Wheat ◽

Root Zone ◽

Wheat Root ◽

Root Surface ◽

Atmospheric Nitrogen ◽

Rhizospheric Soil ◽

Chain Reaction ◽

Wheat Roots ◽

Chlorophyll A And B ◽

Polymerase Chain

Aim. To study the specifi cities of complex inoculation of spring wheat roots with the bacteria of Azospirillum genus and Chaetomium cochliodes Palliser 3250, and the isolation of bacteria of Azospirillum genus, capable of fi xing atmospheric nitrogen, from the rhizospheric soil, washed-off roots and histoshere. Materials and meth- ods. The phenotypic features of the selected bacteria were identifi ed according to Bergi key. The molecular the polymerase chain reaction and genetic analysis was used for the identifi cation the bacteria. Results. It has been demonstrated that during the introduction into the root system of spring wheat the strain of A. brasilensе 102 actively colonizes rhizospheric soil, root surface and is capable of penetrating into the inner plant tissues. Conclusions. The soil ascomucete of C. cochliodes 3250 promotes better settling down of Azospirillum cells in spring wheat root zone, especially in plant histosphere which induces the increase in the content of chlorophyll a and b in the leaves and yield of the crop.

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Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences.

Journal of Bacteriology ◽

10.1128/jb.177.5.1414-1417.1995 ◽

1995 ◽

Vol 177 (5) ◽

pp. 1414-1417 ◽

Cited By ~ 274

Author(s):

T Ueda ◽

Y Suga ◽

N Yahiro ◽

T Matsuguchi

Keyword(s):

Bacterial Diversity ◽

Nifh Gene ◽

Evolutionary Analysis ◽

Gene Sequences ◽

Rice Roots ◽

Molecular Evolutionary Analysis

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The effect of psychrotrophic bacteria isolated from the root zone of winter wheat on selected biotic and abiotic factors

Journal of Plant Protection Research ◽

10.2478/jppr-2014-0061 ◽

2014 ◽

Vol 54 (4) ◽

pp. 407-413 ◽

Cited By ~ 2

Author(s):

Sebastian Wojciech Przemieniecki ◽

Tomasz Paweł Kurowski ◽

Karol Korzekwa ◽

Anna Karwowska

Keyword(s):

Polymerase Chain Reaction ◽

Winter Wheat ◽

Root Zone ◽

Abiotic Factors ◽

Pathogenic Fungi ◽

Growth And Yield ◽

Chain Reaction ◽

Psychrotrophic Bacteria ◽

Rapd Pcr ◽

Polymerase Chain

Abstract The roots of winter wheat plants, cv. Mikon, grown in 45-year monoculture, were analysed in the study. Twenty-two bacterial isolates obtained from the rhizosphere, rhizoplane, and endorhizosphere that were capable of growth at 8°C and at 28°C, were selected for further analysis. The isolated psychrotrophs accounted for 25% of all bacteria present in the wheat rhizosphere and capable of growth at 8°C. Psychrotrophic bacteria were analysed at a temperature of 10°C and 28°C to determine their ability to inhibit the growth of pathogenic fungi, solubilise mineral phosphates, and to determine their ability to degrade chitin and cellulose. Similarity between the isolates was determined by Enterobacterial Repetitive Intergenic Consensus – Polymerase Chain Reaction (ERIC-PCR) and Random Amplification of Polymorphic DNA – Polymerase Chain Reaction (RAPD-PCR). The majority of isolated psychrotrophs inhibited the growth of pathogenic fungi and solubilised mineral phosphates at both incubation temperatures. Psychrotrophic bacteria exerted a two-fold stronger inhibitory effect on mycelial growth at 10°C than at 28°C. The growth of Fusarium culmorum and F. oxysporum was inhibited to the highest extent at 10°C and at 28°C, respectively. Phosphate solubilisation rates were higher at 28°C, particularly in the rhizosphere. Regardless of temperature, the bacteria exhibited low chitin-degrading potential, and none of the isolates was capable of degrading cellulose. A high similarity between the selected psychrotrophs was revealed by ERIC-PCR and RAPD- -PCR analyses. Based on RAPD-PCR, the analysed population was divided into a group of isolates obtained from the rhizosphere, and two groups comprising representatives of both the rhizoplane and the endorhizosphere. Due to their ability to grow over a wide temperature range and increase phosphorus availability to plants, and their antagonism against pathogens, psychrotrophic bacteria can be used to improve the growth and yield of cereal crops.

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Laporan Pertama di Sulawesi Selatan: Karakter Morfologi dan Molekuler Nematoda Puru Akar yang Berasosiasi dengan Akar Padi di Kabupaten Wajo, Sulawesi Selatan

Jurnal Perlindungan Tanaman Indonesia ◽

10.22146/jpti.33108 ◽

2018 ◽

Vol 22 (1) ◽

pp. 58

Author(s):

Hishar Mirsam ◽

Fitrianingrum Kurniawati

Keyword(s):

Polymerase Chain Reaction ◽

Nematode Species ◽

Morphological Characters ◽

Rice Root ◽

Nucleotide Sequence Analysis ◽

Morphological Identification ◽

Root Knot Nematode ◽

Chain Reaction ◽

Meloidogyne Graminicola ◽

Polymerase Chain

Root Knot Nematode (RKN) is one of the most important cosmopolite parasitic nematode species. Reports on RKN associated with rice root in Indonesia are still limited in West Java and Yogyakarta. This study aimed to identify the RKN associated with rice root in Sub-district of Bola, District of Wajo, South Sulawesi, based on morphological and molecular characters. Sampling was carried out by purposive method based on specific criteria of sample, i.e. root knot. Identification of root knot nematode (RKN) infestation in field was done by observing the primary and secondary symptoms. Morphological identification was carried out based on characters of juvenile 2 and the female perineal pattern. Molecular identification was based on amplification of r-DNA by polymerase chain reaction technique using primers rDNA2 and rDNA 1.58s. RKN were detected associated with the incidence of root knot in rice plant. RKN was identified as Meloidogyne graminicola based on morphological characters of juvenile 2 and the female perineal pattern. PCR using primer rDNA 2 / rDNA 1.58s successfully amplified a DNA band of RKN of ± 500 bp. Nucleotide sequence analysis showed that RKN isolated from Wajo was closely related to M. graminicola isolated from Nepal, China, India, Madagascar, and USA with homology of 98.1–100.00%. IntisariNematoda puru akar (NPA) merupakan salah satu jenis nematoda parasit penting yang bersifat kosmopolit. Laporan NPA yang berasosiasi dengan akar tanaman padi di Indonesia masih terbatas di Jawa Barat dan Yogyakarta. Penelitian ini bertujuan mengidentifikasi NPA yang berasosiasi dengan akar tanaman padi di Kabupaten Wajo, Sulawesi Selatan berdasarkan karakter morfologi dan molekuler. Pengambilan sampel dilakukan secara purposif dengan memilih sampel berdasarkan pada kriteria gejala spesifik penyakit puru akar. Identifikasi serangan dilakukan dengan mengamati gejala primer dan gejala sekunder. Identifikasi morfologi dilakukan dengan pengamatan karakter morfologi juvenil 2 dan pola perineal NPA betina.Identifikasi molekuler dilakukan dengan teknik polymerase chain reaction (PCR) untuk mengamplifikasi wilayah internal transcribed spacer (ITS) ribosomal DNA (rDNA) menggunakan pasangan primer rDNA2 dan rDNA1.58s. NPA ditemukan berasosiasi dengan akar tanaman padi yang memperlihatkan gejala puru akar. NPA diidentifikasi sebagai Meloidogyne graminicola berdasarkan karakter morfologi juvenil 2 dan pola perineal NPA betina. PCR menggunakan primer rDNA2/ rDNA1.58s berhasil mengamplifikasi pita DNA NPA dengan ukuran sekitar 500 bp. Analisis runutan nukleotida menunjukkan isolat NPA asal Wajo-Indonesia memiliki tingkat kekerabatan yang sangat dekat dengan isolat M. graminicola asal Nepal, Cina, India, Madagaskar, dan Amerika Serikat dengan nilai homologi berkisar 98,1–100,0%.

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Predominance of hydrogen-utilizing bacteria among N2-fixing bacteria in wetland rice roots

Canadian Journal of Microbiology ◽

10.1139/m82-157 ◽

1982 ◽

Vol 28 (9) ◽

pp. 1051-1054 ◽

Cited By ~ 9

Author(s):

I. Watanabe ◽

W. L. Barraquio ◽

Maria Luisa Daroy

Keyword(s):

Rhizosphere Soil ◽

Heterotrophic Bacteria ◽

Rice Root ◽

Wetland Rice ◽

Hydrogenase Activity ◽

Wetland Soil ◽

Uptake Hydrogenase ◽

Autotrophic Growth ◽

Rice Roots ◽

Soil Rhizosphere

Heterotrophic bacteria were isolated from wetland soil, rhizosphere soil, root and basal shoot of wetland rice, dryland soil, and root of dryland rice. The isolates were tested for N2-fixing activity and the ability to grow autotrophically under H2 + CO2 + O2. N2-fixing bacteria capable of autotrophic growth were found almost exclusively from the rhizosphere and the root of wetland rice. In another experiment, all N2-fixing bacteria isolated from wetland rice root had uptake hydrogenase activity. These findings indicate the predominance of hydrogen-utilizing bacteria among N2-fixing bacteria from wetland rice roots.

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Comparison of Mitochondrial DNA in Wolves and Coyotes in the Northern Rockies using the Polymerase Chain Reaction

The UW National Parks Service Research Station Annual Reports ◽

10.13001/uwnpsrc.1994.3221 ◽

1994 ◽

Vol 18 ◽

pp. 156-159

Author(s):

Ernest Vyse

Keyword(s):

Polymerase Chain Reaction ◽

Mitochondrial Dna ◽

National Park ◽

Genetic Basis ◽

Gene Activation ◽

Evolutionary Analysis ◽

Chain Reaction ◽

Variable Regions ◽

Conserved Regions ◽

Polymerase Chain

The purpose of this study was the comparison of mitochondrial DNA (mtDNA) sequences from Northern Montana and Minnesota wolves to the extinct wolves of Yellowstone National Park (YNP). This comparison was intended to provide a genetic basis to identify potential wolf stocks for introduction into YNP. Unfortunately the only extinct YNP specimens available were dried tanned skins from the Smithsonian Museum of Natural History and YNP. The nucleic acids extracted from these skins was so badly degraded that no amplification of mtDNA was possible; so the critical comparisons could not be made. The control region of the mtDNA is 1100-1200 nucleotides long in vertebrates, contains sequences that control replication and gene activation and in terms of evolutionary analysis has the desired feature of both variable and conserved regions. The variable regions provide the genetic diversity that is the basis of taxonomic and evolutionary studies while the conserved regions provide the ability to synthesize short complementary oligonucleotides to prime the polymerase chain reaction (PCR) in the organisms studies.

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High performance Nanogold™-in situ hybridzation and its use in the detection of hybridized and PCR-amplified microscopical preparations

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166944 ◽

1996 ◽

Vol 54 ◽

pp. 896-897

Author(s):

G. W. Hacker ◽

I. Zehbe ◽

J. Hainfeld ◽

A.-H. Graf ◽

C. Hauser-Kronberger ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Messenger Rna ◽

High Performance ◽

Detection System ◽

Direct Methods ◽

Genetic Alterations ◽

Chain Reaction ◽

Protein Encoding ◽

Polymerase Chain

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

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