• Journal of Microbiology
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• v.38 no.1
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• pp.11-17
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• 2000

A new method was developed for the rapid analysis of diverse bacterial species in the natural environment. Our method is based on PCR-single-strands-conformation polymorphism (PCR-SSCP) and selective isolation technique of single-stranded DNA. Variable V3 fragments of 16S rDNA were amplified by PCR with bacterial 16S rDNA primers, where one of the primers was biotinylated at the 5'-end. The biotinylated strands of the PCR products were selectively isolated by using streptavidin paramagnetic particles and a magnetic stand, to prevent SSCP analysis producing heteroduplexes from heterogeneous DNA samples. The selected strands were separated by electrophoresis on a polyacrylamide gel, and detected by silver staining. Analysis of PCR products from 8 bacterial strains demonstrated their characteristic DNA band patterns. In addition, changes in the structure of the bacterial community and species diversity in the microcosm treated with phenol could be monitored. After 3 weeks of incubation, phenol and its intermediate, 2-hydroxy-muconic-semialdehyde, were degraded by indigenous bacteria. These dominating bacterial populations were identified as strong bands on an SSCP gel. Therefore, this study provides useful tools for microbial community analysis of natural habitats.

• The Plant Pathology Journal
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• v.34 no.2
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• pp.143-149
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• 2018

Flowers of kiwifruit are morphologically hermaphroditic and survivable binucleate pollen is produced by the male flowers. In this study, we investigated microbial diversity in kiwifruit pollens by analyzing amplicon sequences of 16S rRNA. Four pollen samples were collected: 'NZ' was imported from New Zealand, 'CN' from China in year of 2014, respectively. 'KR13' and 'KR14' were collected in 2013' and 2014' in South Korea. Most of the identified bacterial phyla in the four different pollens were Proteobacteria, Actinobacteria and Firmicutes. However, the imported and the domestic pollen samples showed different aspects of microbial community structures. The domestic pollens had more diverse in diversity than the imported samples. Among top 20 OTUs, Pseudomonas spp. was the most dominant specie. Interestingly, a bacterial pathogen of kiwifruit canker, Pseudomonas syringae pv. actinidiae was detected in 'NZ' by the specific PCR. This study provides insights microbial distribution and community structure information in kiwifruit pollen.

• Ocean and Polar Research
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• v.39 no.4
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• pp.245-255
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• 2017

There are a number of pieces of evidences that suggest a link between marine diatoms and microorganisms, but knowledge about related microbial communities is greatly lacking. The present study investigated the microbial community structures related to the growth of the marine diatom Cyclotella meneghiniana. We collected free-living bacteria (FLB) and particle-associated bacteria (PAB) at each growth stage (e.g., lag, exponential, stationary and death) of the diatom, and analyzed their bacterial 16S rDNA using pyrosequencing. Metagenomics analysis showed that community structures of FLB and PAB differed considerably with the progress of growth stages. FLB showed higher diversity than PAB, but variation in the different growth stages of C. meneghiniana was more evident in PAB. The proportion of the genus Hoeflea, belonging to the order Rhizobiales, was dominant in both FLB and PAB, and it gradually increased with the growth of C. meneghiniana. However, Enhydrobacter clade tended to considerably decrease in PAB. In addition, Marinobacter decreased steadily in FLB, but first increased and then decreased in PAB. These results suggest that Hoeflea, Enhydrobacter, and Marinobacter may be closely related to the growth of diatom C. meneghiniana.

• The Plant Pathology Journal
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• v.37 no.4
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• pp.396-403
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• 2021

Fire blight disease, caused by Erwinia amylovora, could damage rosaceous plants such as apples, pears, and raspberries. In this study, we designed to understand how E. amylovora affected other bacterial communities on apple rhizosphere; twig and fruit endosphere; and leaf, and fruit episphere. Limited studies on the understanding of the microbial community of apples and changes the community structure by occurrence of the fire blight disease were conducted. As result of these experiments, the infected trees had low species richness and operational taxonomic unit diversity when compared to healthy trees. Rhizospheric bacterial communities were stable regardless of infection. But the communities in endosphere and episphere were significanlty affected by E. amylovora infection. We also found that several metabolic pathways differ significantly between infected and healthy trees. In particular, we observed differences in sugar metabolites. The finding provides that sucrose metabolites are important for colonization of E. amylovora in host tissue. Our results provide fundamental information on the microbial community structures between E. amylovora infected and uninfected trees, which will contribute to developing novel control strategies for the fire blight disease.

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.21-29
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• 2010

The phenolic compounds are a major contaminant class often found in industrial wastewaters and the biological treatment is an alternative tool commonly employed for their removal. In this sense, monitoring microbial community dynamics is crucial for a successful wastewater treatment. This work aimed to monitor the structure and activity of the bacterial community during the operation of a laboratory-scale continuous submerged membrane bioreactor (SMBR), using PCR and RT-PCR followed by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA libraries. Multivariate analyses carried out using DGGE profiles showed significant changes in the total and metabolically active dominant community members during the 4-week treatment period, explained mainly by phenol and ammonium input. Gene libraries were assembled using 16S rDNA and 16S rRNA PCR products from the fourth week of treatment. Sequencing and phylogenetic analyses of clones from the 16S rDNA library revealed a high diversity of taxa for the total bacterial community, with predominance of Thauera genus (ca. 50%). On the other hand, a lower diversity was found for metabolically active bacteria, which were mostly represented by members of Betaproteobacteria (Thauera and Comamonas), suggesting that these groups have a relevant role in the phenol degradation during the final phase of the SMBR operation.

• Journal of Microbiology and Biotechnology
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• v.13 no.2
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• pp.236-242
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• 2003

A culture-independent and -dependent survey of the bacterial community structure in the rhizosphere and soil samples from hot pepper plants was conducted using 16S rDNA clone library and FAME analyses. Out of the 78 clones sequenced, 56% belonged to Proteobacteria, 4% to high G+C Gram- positive group, 3% to Cytophyga-Flexibacter-Bacreroides, and 32% could not be grouped with any known taxonomic division. Among the 127 FAME isolates identified, 66% belonged to low G+C Gram-positive bacteria (Baciilus spp.) and 26% to high G+C Gram-positive bacteria. In a cluster analysis, the results for both methods were found to be strikingly dissimilar. The current study is the first comparative study of FAME and 165 rDNA clonal analyses performed on the same set of soil, rhizosphere soil, and root samples.

• The Plant Pathology Journal
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• v.32 no.2
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• pp.136-144
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• 2016

Pseudomonas fluorescens pc78 is an effective biocontrol agent for soil-borne fungal diseases. We previously constructed a P43-gfp tagged biocontrol bacteria P. fluorescens pc78-48 to investigate bacterial traits in natural ecosystem and the environmental risk of genetically modified biocontrol bacteria in tomato rhizosphere. Fluctuation of culturable bacteria profile, microbial community structure, and potential horizontal gene transfer was investigated over time after the bacteria treatment to the tomato rhizosphere. Tagged gene transfer to other organisms such as tomato plants and bacteria cultured on various media was examined by polymerase chain reaction, using gene specific primers. Transfer of chromosomally integrated P43-gfp from pc78 to other organisms was not apparent. Population and colony types of culturable bacteria were not significantly affected by the introduction of P. fluorescens pc78 or pc78-48 into tomato rhizosphere. Additionally, terminal restriction fragment length polymorphism profiles were investigated to estimate the influence on the microbial community structure in tomato rhizosphere between non-treated and pc78-48-treated samples. Interestingly, rhizosphere soil treated with strain pc78-48 exhibited a significantly different bacterial community structure compared to that of non-treated rhizosphere soil. Our results suggest that biocontrol bacteria treatment influences microbial community in tomato rhizosphere, while the chromosomally modified biocontrol bacteria may not pose any specific environmental risk in terms of gene transfer.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.287-295
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• 2005

In order to compare bacteria] community structure and diversity in activated sludges, terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified 16s rDNAs was analyzed for 31 domestic and industrial wastewater treatment plants (WTPs). Regardless of the characteristics of the wastewaters, the bacteria] community structures of activated sludges appeared diverse and complex. In particular, activated sludges in domestic WTPs contained higher bacterial diversity than those in industrial WTPs. It was also found that terminal restriction fragment (T-RF) profiles derived from domestic WTPs were very similar with each other, although activated sludges were collected from different plants at different locations. Interestingly, activated sludges of a WTP where restaurant and toilet sewages of a company were managed showed a bacterial community structure similar to that of domestic WTPs. Activated sludges in leather industria] WTPs also showed a high similarity. However, other wastewaters possessed different bacterial communities, so that overall similarity was as low as about $30\%$. Since activated sludges from WTPs for domestic wastewaters and a company sewage appeared to hold similar bacterial communities, it was necessary to confirm if similar wastewaters induce a similar bacterial community. To answer this question, analysis of T-RFs for activated sludges, taken from another 12 domestic WTPs, was conducted by using a 6­FAM$^{TM}$-Iabeled primer and an automated DNA sequencer for higher sensitivity. Among 12 samples, it was again found that T-RF profiles of activated sludges from Yongin, Sungnam, Suwon, and Tancheon domestic WTPs in Kyonggi-do were very similar with each other. On the other hand, T-RF profiles of activated sludges from Shihwa and Ansan WTPs were quite different from each other. It was thought that this deviation was caused by wastewaters, since Ansan and Shihwa WTPs receive both domestic and industrial wastewaters. From these results, it was tentatively concluded that similar bacterial communities might be developed in activated sludges, if WTPs treat similar wastewaters.

• Korean Journal of Soil Science and Fertilizer
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• v.46 no.6
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• pp.585-592
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• 2013

To estimate the effect of long-term fertilization on metabolically active bacterial communities in a rice field, RNA was extracted from endosphere (rice root), rhizosphere, and bulk soil that had been subjected to different fertilization regimes for 59 years, and the 16S rRNAs were analyzed using the pyrosequencing method. The richness and diversity of metabolically active bacteria were higher in bulk soil than in the endosphere and rhizosphere, and showed no significant difference between non-fertilized and fertilized plots. Weighted UniFrac analysis showed that each compartment had characteristic bacterial communities and that the effect of long-term fertilization on the structure of bacterial community was more pronounced in bulk soil than in the endosphere and rhizosphere. The 16S rRNAs affiliated with Alphaproteobacteria and Firmicutes were more abundant in the endosphere than in bulk soil while those affiliated with Chloroflexi and Acidobacteria were more abundant in bulk soil than in the endosphere. Several dominant operational taxonomic units (clustered at a 97% similarity cut-off) showed different frequencies between non-fertilized and fertilized plots, suggesting that the fertilization affected their activities in the rice field.

• Journal of Microbiology and Biotechnology
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• v.26 no.1
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• pp.89-98
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• 2016

Endophytes play an important role in the growth and development of the host. However, the study of endophytes is mostly focused on plants, and reports on bacteria associated with fungi are relatively rare. We studied the bacteria associated with fruiting bodies of Tricholoma matsutake picked from seven main T. matsutake-producing areas in Sichuan, China, by barcoded pyrosequencing. About 8,272 reads were obtained per sample, representing 40 phyla, 103 classes, and 495 genera of bacteria and archaea, and 361-797 operational taxonomic units were observed at a 97% similarity level. The bacterial community was always both more abundant and more diverse than the archaeal community. UniFrac analysis showed there were some difference of bacterial communities among the samples sites. Three bacterial phyla, Proteobacteria, Bacteroidetes, and Firmicutes, were dominant in all samples. Correlation analysis showed there was a significant correlation between some soil properties and bacterial community associated with T. matsutake. This study demonstrated that the bacteria associated with T. matsutake fruiting bodies were diversified. Among these bacteria, we may find some strains that can promote the growth of T. matsutake.