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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.51 No.5 pp.9-16

Revealed the Status of Microbial Diversity and Structure in Soil and Rhizosphere of Endangered Plant Species Cypripedium japonicum

Hye Sun Kim1, Geun-Hye Gang2, Eun-Hee Park2, Youn-Sig Kwak1*
1Division of Applied Life Science(BK21 Plus) and Institute of Agriculture and life Science, Gyeongsang National University, Jinju, 52828, Korea
2Species Restoration Technology Institute, Korea National Park Service, Muju, 55557, Korea
Corresponding author: Youn-Sig Kwak
20170201 20170318 20170529


The Cypripedium japonicum is a species of orchid, distributed mainly in the temperate regions of the Northern Hemisphere. In Korea, C. japonicum is an extremely rare species and class I endangered plant. However, little is known about the plant-microbe interaction and its ecology, especially rhizosphere bacterial communities in terms of structure and diversity in C. japonicum. In this study, the diversity of bacterial communities was investigated in C. japonicum rhizosphere and bulk soil. Proteobacteria, Acidobacteria and Actinobacteria were the most abundant groups in both rhizosphere and bulk soil. Significantly Proteobacteria and Actinobacteria abundantly inhabited in the rhizosphere when compared with the bulk soil. Conversely, significantly more unclassified bacteria were detected in the rhizosphere soil than in the bulk soil. This work increases our understanding of the rhizosphere bacterial diversity on class I endangered plant, C. japonicum.



    Orchid is one of the largest family in plant kingdom, more than 25,000 species were recorded (Cribb et al., 2003). Among them, Genus Cypripedium are distribution 40 species worldwide and have been reported 4 species(Cypripedium japonicum, C. macranthum, C. guttatum, and C. calceolus) in Korea(Sim et al., 2010). C. japonicum are distributed in China, Taiwan, Japan and Korea, Cypripedium is distributed in Korea, Northern China, Japan and Eastern Europe (Cash, 1991). However, populations of these plants have been decreasing by deforestation, global warming and destruction of habitats and ecosystems(Smith & Read, 2008). Taxonomical and functional structure of soil bacterial communities are influenced by several biotic and abiotic factors including soil characteristics, climate variables, plant species and phenology, and the interactions established with other soil organisms. Similarly, soil characteristics, particularly pH and fertility status, have shown to correlate with overall bacterial diversity(Dunbar et al., 1999; Acosta- Martinez et al., 2008; Fulthorpe et al., 2008). In addition, plants select specific microbial populations from the soil reservoir through the release of root exudates(Haichar et al., 2008; Berg & Smalla, 2009). These exudates play complex roles, because they influence the physicochemical characteristics of the bulk soil(Ranger & Turpault, 1999) and can also be used as growth substrates or signaling molecules by the soil bacterial communities(Shaw et al., 2006). Collectively, these modifications allow the maintenance of a dynamic and nutrient-rich niche around the root tips, also known as rhizosphere, where the activity of specific bacterial communities enhanced to compare with that of the bacterial communities from the bulk soil. Several reports based on microcosms and field experiments with natural or genetically modified plants have highlighted a shift of the bacterial communities colonizing the rhizosphere compared with that of the surrounding soil. These studies showed an enrichment of specific taxonomic or functional groups in the rhizosphere(Oger et al., 2004; Frey-Klett et al., 2005). Bacterial communities colonizing on rhizosphere have been shown to help the plants in several ways, including supply of inorganic nutrients(Uroz et al., 2009), enhancement of nitrogen uptake(Cocking, 2003) and protection against pathogens. The rhizosphere microbiomes have been studied using fingerprinting methods, cloning-sequencing procedures(Smalla et al., 2007). These methods were used to compare the bacterial community patterns from several soil samples collected from different ecological environments such as the rhizosphere niche and the surrounding soil (Smalla et al., 2007). More recently, high-density 16S rRNA microarray analyses were also developed to compare the bacterial communities inhabiting the rhizosphere and the surrounding soil of cereals (Sanguin et al., 2006; 2008; De Angelis et al., 2008) and for the evaluation of the potential impact of global warming on pristine systems(Yergeau et al., 2009). Panwar & Vyas(2002) reported beneficial effects of rhizobiomes on endangered plant species and proposed use of the beneficial microbes in conservation of these endangered multipurpose tree species in the Indian desert.

    In this study, the composition of bacterial communities inhabiting on the C. japonicum rhizosphere and bulk soil were investigated using pyrosequencing. Number of operational taxonomic units(OTUs), species richness, and the differences occurring between the rhizosphere and bulk soils were investigated. The results may contribute to gain knowledge of C. japonicum ecology, microbe interaction and apply on protection and restoration of endangered plant.

    Materials and Methods

    1.Field collection of rhizosphere and bulk soil of C. japonicum

    The sampling site was a natural habitat protection area in the Deogyu National Park, South Korea. In 2015, the 2 sampling sites were selected based on their location relative to C. japonicum and rhizosphere soil, bulk soil samples were collected. Subsequently, the two soil samples were collected with four replications in each region were used for further experiments.

    2.Soil DNA extraction, amplicon preparation and pyrosequencing analysis

    DNA was extracted using a Soil DNA Isolation Kit(MP biomedicals, CA, USA) according to the manuals. Total 8 soil samples(4 for rhizosphere and 4 for bulk soils) were used to extract DNA. Extracted DNA was stored at -80°C until used. The bacterial 16S rRNA gene was amplified from each sample using the 27F(5′-agagtttgatcmtggctcag-3′) and 1492R (5′-ggytaccttgttacgactt-3′) primers. Pyrosequencing was performed by ChunLab, Inc.(Seoul, South Korea) using a Roche/454 GS FLX Titanium platform. Sequences were processed and analyzed according to the bioinformatics procedures described by Chun et al.(2010) and Singh et al.(2012). Raw sequencing reads from different soil samples were separated by unique barcode sequences. Sequences with short lengths(<300 bp) or including >2 ambiguous bases (Ns) were removed before analysis. Primer, linker, and barcode sites were then trimmed by pairwise alignment. Nonspecific PCR amplicons that showed no matches in the 16S rRNA gene database using the EzTaxon database were discarded. All sequence reads were additionally screened for chimeras using the BLAST program. Taxonomic assignment was carried out by comparing the sequence reads against the EzTaxon-e database, using a combination of the initial BLAST-based searches and additional pairwise similarity comparisons. The following criteria were applied for the taxonomic assignment of each read(x = distance values): species(x 0.03), genus(0.03<x 0.05), family(0.05<x 0.1), order(0.1<x 0.15), class(0.15<x 0.2), and phylum(0.2<x 0.25)(Stackebrandt & Goebel 1994; Sait et al. 2002; Schloss & Handelsman 2004). If the distance was greater than the cutoff value, the read was assigned to an unclassified group. If the sequence cluster could not be identified with a valid name, the accession number of the GenBank sequence entry sharing the highest sequence similarity with the sequence cluster was used as a provisional name. All statistical analyses of the bacterial communities were carried out with CLcommunity software(Chunlab, Inc., Seoul, South Korea). The operational taxonomic units(OTUs) were defined with the CD-HIT program at 3% sequence dissimilarity.

    Results and Discussion

    In all, 1,300(bulk) to 1,750(rhizosphere) OTUs per soil sample were obtained at a 99% similarity level. The richness of bacterial OTUs differed, depending on the type of sampling sites. The species richness of each sample indicated that the rhizosphere soil had a greater microbial community than bulk soil samples (data not shown). Each sequence of bacterial rRNA gene was classified from phylum down to the genus level using CLcommunity software(Chunlab, Inc., Seoul, South Korea). Taxonomic composition analysis was conducted at the phylum level(Fig. 1). Rhizosphere soil sample has Proteobacteria 49.9%, Acidobacteria 20.5%, Actinobacteria 12.4%, Pianctomycetes 3.4%, Bacteroidetes 3.0%, Chloroflexi 2.8%, Nitrospirae 2.2%, and Gemmatimonadetes 1.5%. Bulk soil sample has Proteobacteria 47.0%, Acidobacteria 28.7%, Actinobacteria 7.0%, Planctomycetes 3.5%, Bacteroidetes 1.4%, Chloroflexi 3.6%, Nitrospirae 2.3%, Firmicutes 1.6%, and Verrucomicrobia 1.2%. The Proteobacteria were the dominant phyla in all soil samples. Similar proportions of these phyla were reported in agricultural and forest soil samples(Roesch et al., 2007; Fulthorpe et al., 2008; Uroz et al., 2010). De Angelis(2008) reported that a significantly larger number of microorganisms were detected in the rhizosphere in comparison with bulk soil. Overall, the rhizosphere and bulk soil seemed to have similar relative abundances across the phylum level (Fig. 1). In addition, Top 20 OTUs was similar in both samples(Table 1, Table 2). The result supported that Proteobacteria were abundance in both rhizosphere and bulk soil microbial. The relative abundance at the class level for Alphaproteobacteria, the most frequently observed phyla in both samples, showed distinct differences between two zones(data not shown). Sampling zones shared the following 12 classes: Alphaproteobateria, Solibacteres, Acidobateria_c, Actinobacteria_c, Betaproteobacteria, Planctomycetacia, Deltaproteobacteria, Gammaproteobacteria, EU686603_c, Rubrobacteria, GU444092_c, and Sphingobacteria. In contrast, Gemmatimonadetes_c, Chloracidobacterium_c, Acidimicrobiia, and Thermoleophilia were not found bulk soil and Verrucomicrobiae, Bacilli, and Ktedonobacteria were not found rhizosphere soil. The Alpharoteobacteria were the dominant class and Bradyrhizobiaceae were the dominant family in the both soils. The result supported that rhizosphere microbes were more diverse than bulk soil. This contrasts with studies of the rhizosphere of grape in which there were significantly more Betaproteobacteria in the rhizosphere than in the bulk soil, and significantly more Alphaproteobacteria in the bulk soil than in rhizosphere(Sanguin et al., 2006; Haichar et al., 2008). Bacterial communities are acknowledged as one of the major components of soil function, playing a key role in niche maintenance(Molina et al., 2000). The taxonomic composition of the sample showed a statistical picture(Fig. 2). The pie chart depicts the taxonomic compositions of phylum ranks simultaneously. Proteobacteria, Acidobacteria, Actinobacteria, Planctomycetes, Chloroflexi, Nitrospirae, Bacteroidetes, Firmicutes, Verrucomicrobia, Gemmatimonadetes, AD3, Armatimonadetes, TM7, Elusimicrobia, WS3, TM6, Cyanobacteria, MATCR, WS5, OP3, OD1, Chlorobi, Spirochaetes, Streptophyta, BRC1, DQ833500_p, 4P 001887_p. The both samples were the dominant Proteobacteria, Acidobacteria, and Actinobacteria. The rhizosphere soil was abundance Proteobacteria and Actinobacteria more than bulk soil. Acidobacteria was more abundance in the bulk soil than rhizosphere soil. To the restoration of endangered plants, it will be made to a variety of endangered plants and more research on the soil of habitat. This paper will be the basis of research for restoration.


    This research was supported by the MOU between Species Restoration Technology Institute, Korea National Park Service and College of Agriculture and Life Science, Gyeongsang National University.



    Comparison of microorganism between the rhizosphere soil and the bulk soil at the phylum level.


    Average composition pie chart presenting the summed composition of all samples. The circle denotes phylum.


    The microbial OTUs, which exist only in rhizosphere soil

    The microbial OTUs, which exist only in bulk soil


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