Community Analysis of Seed-Associated Microbes in Forage Crops using Culture-Independent Methods

Ikeda, Seishi; Fuji, Shin Ichi; Sato, Toshiro; Ytow, Nozomi; Ezura, Hiroshi; Minamisawa, Kiwamu; Fujimura, Tatsuhito

doi:10.1264/jsme2.21.112

Cited by 22 publications

(17 citation statements)

References 49 publications

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“…RISA amplicons which were consistent among triplicate samples were selected and extracted from the gel as described in a previous report from our laboratory 17) . The cloning and sequencing of these amplicons were carried out as described in the previous section.…”

Section: Ribosomal Intergenic Spacer Analysismentioning

confidence: 99%

Evaluation of the Effects of Different Additives in Improving the DNA Extraction Yield and Quality from Andosol

et al. 2008

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A series of additives were evaluated for their effects on improving the yield and quality of DNA extracted from recalcitrant soils. Levels of possible DNA contaminants in these supplements were also assessed. Three of the additives (skim milk, casein, and RNA) were shown to be effective in improving the stable extraction of DNA from recalcitrant samples of Andosol. However, whereas skim milk appeared to be the most effective additive for this purpose, our data indicated that this commercially sourced product contained considerable amounts of contaminant DNA (30 to 40 µg/g skim milk). A ribosomal intergenic spacer analysis (RISA) revealed the consistent contamination of different batches of this product with DNA of several species of both eukaryotes (cattle and protists) and prokaryotes. In particular, thermophilic bacteria such as Geobacillus and Anoxybacillus were identified in the sequenced PCR amplicons from skim milk. The results of the RISA clearly also indicated that the impact of contaminated DNA on the analysis of a microbial community could be significant when skim milk is used for extracting DNA from a recalcitrant soil. In contrast, only a trace amount of contaminating DNA was evident in casein and none was detected in the RNA examined in the present study.

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Section: Ribosomal Intergenic Spacer Analysismentioning

confidence: 99%

Evaluation of the Effects of Different Additives in Improving the DNA Extraction Yield and Quality from Andosol

et al. 2008

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“…The ribosomal intergenic spacer analysis (RISA) was carried out using modifications previously reported by Ikeda et al 13,15) . Briefly, DNA was directly extracted using a FastDNA Spin kit for soil (Applied Biosystem, Carlsbad, CA, USA) with bead-beating to disrupt the microbial cells.…”

Section: Ribosomal Intergenic Spacer Analysis (Risa)mentioning

confidence: 99%

“…The primer set was ITSF/ITSReub, which targeted the end of the 16S rRNA gene and the beginning of the 23S rRNA gene for bacterial RISA 5) . The PCR temperature program and gel electrophoresis were as previously described 15) . MM-CFTFL 50-1000 MapMarker (BioVentures, Inc. Murfreesboro, TN, USA) was used as a size marker.…”

Section: Ribosomal Intergenic Spacer Analysis (Risa)mentioning

confidence: 99%

Broad Distribution and Phylogeny of Anaerobic Endophytes of Cluster XIVa Clostridia in Plant Species Including Crops

et al. 2008

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Endophytic clostridia present on various plants as obligate anaerobes were surveyed by terminal restriction fragment length polymorphism (TRFLP) analysis specific to the clostridial 16S rRNA gene. Endophytic clostridia were detected in 10 plant types: sugarcane, cultivated rice, corn, tobacco, soybean, bermuda grass, tall fescue, and three mangrove species. Phylogenetically, cluster XIVa clostridia were detected more frequently than cluster I clostridia in aerial parts. Isolation of clostridia from surface-sterilized sugarcane stem validated the TRFLP results. Plant-derived clostridia occupied two unique phylogenetic positions (groups I and II) within cluster XIVa. Most of cluster XIVa clostridia from other sources (e.g., human, animal, and insect intestines) were located outside these groups. Thus two unique groups of cluster XIVa clostridia are widely distributed in plants, including crops. In field-grown soybeans, TRFLP analysis revealed clostridia only in a non-nodulating mutant. Ribosomal intergenic spacer analysis (RISA) showed that the bacterial community in soybean shoot depended partly on the soybean nodulation genotype.Key words: Clostridium, cluster XIVa, endophyte, sugarcane, soybean Endophytes are microorganisms that can colonize living plant tissues without causing any apparent damage to the host 3,23,24,35) . Several diazotrophs have been isolated and characterized as nitrogen-fixing endophytes in plants, including Gluconacetobacter diazotrophicus 3,22) , Herbaspirillum spp. 9,33,38,48) , and Azoarcus sp.19,31) . Novel anaerobic endophytic clostridia and their anaerobic nitrogen-fixing consortium (ANFICO) were recently isolated and characterized in grasses during attempt to overcome a problem with culturing nitrogen-fixing microbes 26) . ANFICO consists of nitrogenfixing clostridia and diverse non-diazotrophic bacteria and expresses nitrogen-fixing activity associated with low oxygen tension and interspecies inducers 26) . Phylogenetic analysis indicated that the endophytic clostridia fell exclusively into clusters XIVa and I, as defined by Collins et al. 8) , and were further subdivided into five groups 26) . Miyamoto et al.27) developed a terminal restriction fragment length polymorphism (TRFLP) detection system specific for these clusters and groups of plant clostridia, and demonstrated that the group II clostridia in cluster XIVa 8)dominated the populations of diazotrophs and clostridia in a gramineous grass, Miscanthus sinensis. Inoculation examinations indicate that endophytic clostridia colonize plant tissues, alleviating the damage caused by salt stress 47) and potentially fixing nitrogen in the plant 34) . Thus, more attention should be paid to the endophytic clostridia in terms of their distribution, phylogeny, and function.Recently, cluster XIVa clostridia have been detected and isolated from human faeces 12,18,28) , animal intestines 2,20,21,25,44) , termite guts 40,41) , soil 7,17,45) , and plants 26,27) using culture-independent and culture-dependent methods. However, the relations...

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“…8) The application of molecular techniques has also accelerated investigation of the community structures of plantassociated bacteria. [9][10][11][12] However, there is a major limitation in the analysis of the community structures targeting the small subunit (SSU) rRNA genes as the barcoding region by cultureindependent molecular techniques; Plant organelle (mitochondria and plastid) genes are simultaneously extracted during the DNA extraction step, then the organelle SSU rRNA genes are easily amplified by PCR with a primer set specific for bacteria. 13−17) In response to this limitation, locked nucleic acid (LNA) oligonucleotides-PCR clamping technique was developed.…”

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Development of LNA oligonucleotide–PCR clamping technique in investigating the community structures of plant-associated bacteria

Ikenaga

Tabuchi

Oyama

et al. 2015

Bioscience, Biotechnology, and Biochemistry

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Simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is major limitation in investigating the community structures of plant-associated bacteria. Although locked nucleic acid (LNA) oligonucleotides was designed to selectively amplify the bacterial small subunit rRNA genes by applying the PCR clamping technique, those for plastids were applicable only for particular plants, while those for mitochondria were available throughout most plants. To widen the applicable range, new LNA oligonucleotides specific for plastids were designed, and the efficacy was investigated. PCR without LNA oligonucleotides predominantly amplified the organelle genes, while bacterial genes were predominantly observed in having applied the LNA oligonucleotides. Denaturing gradient gel electrophoresis (DGGE) analysis displayed additional bacterial DGGE bands, the amplicons of which were prepared using the LNA oligonucleotides. Thus, new designed LNA oligonucleotides specific for plastids were effective and have widened the scope in investigating the community structures of plant-associated bacteria.

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Community Analysis of Seed-Associated Microbes in Forage Crops using Culture-Independent Methods

Cited by 22 publications

References 49 publications

Evaluation of the Effects of Different Additives in Improving the DNA Extraction Yield and Quality from Andosol

Evaluation of the Effects of Different Additives in Improving the DNA Extraction Yield and Quality from Andosol

Broad Distribution and Phylogeny of Anaerobic Endophytes of Cluster XIVa Clostridia in Plant Species Including Crops

Development of LNA oligonucleotide–PCR clamping technique in investigating the community structures of plant-associated bacteria

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