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. 2019 Jul 22;9(8):300. doi: 10.1007/s13205-019-1838-x

Metagenomic analysis of bacterial endophyte community structure and functions in Panax ginseng at different ages

Chi Eun Hong 1, Jang Uk Kim 1, Jung Woo Lee 1, Kyong Hwan Bang 1, Ick Hyun Jo 1,✉
PMCID: PMC6646489  PMID: 31355109

Abstract

This study investigated the root-associated bacterial endophytes of Panax ginseng at different ages by shotgun metagenomic analysis. After mapping metagenome data to the complete ginseng genome to identify unmapped sequences, we predicted the structure and functions of ginseng bacterial endophytes by metagenomic rapid annotation using subsystems technology analysis. While Proteobacteria and Actinobacteria were the predominant phyla in all samples (2–6-year-old roots), class Alphaproteobacteria was most abundant in 3-, 4-, and 5-year-old plants. We found that 3-year-old P. ginseng had a 0.66% unmapped rate against the whole ginseng genome and showed the greatest diversity of endophytic bacteria (α diversity = 299). Prediction of endophytic bacterial functions at different ages by SEED subsystem analysis revealed that siderophore and auxin-related traits—which are known to promote plant growth—were most highly represented in 3-year-old plants. This was supported by a gene frequency analysis of plant growth-promoting genes, including those responsible for solubilization of phosphate and nitrogen metabolism, using BLASTn. These results suggest that endophytic bacteria of the P. ginseng root affect plant growth. Furthermore, the isolation and purification of plant growth-promoting endophytes identified in this study could promote sustainable cultivation of ginseng in the future.

Keywords: Panax ginseng, Bacterial endophyte, Shotgun metagenomics, Siderophore

Introduction

Plants are subjected to various stresses when planted in soil as they cannot be moved during the growth period. As such, plants have evolved various survival strategies to alleviate stress through interactions with microorganisms including bacteria, fungi, and viruses that generally exist symbiotically with plants. Particularly, endophytic bacteria have functions that are important for plant physiology such as antibiotic activity, stimulation of plant hormone production, and enhancement of immunity, all of which promote growth and protect plants from biotic and abiotic stresses (Jasim et al. 2014). An endophyte is generally defined as a microorganism that exists in a mutual or commensal relationship with a plant, spending some or all of its life within the host (Hardoim et al. 2015). Endophytes, thus, secure a stable habitat and nutrients from plants and can exert more lasting effects than other rhizobacteria (Hong et al. 2015).

Most studies to date on endophytic bacteria have been carried out using culture-dependent methods. In fact, very few microorganisms are culture dependent (1–2%) and most are non-culturable (Amann et al. 1995). The metagenome is the total genetic information of microorganisms in a specific environment; metagenomics is the study of this information. Recent advances in next-generation sequencing (NGS) technology have enabled metagenome analyses of soil, rhizospheres, and endophytes (Akinsanya et al. 2015; Campisano et al. 2014; Correa-Galeote et al. 2018; Sengupta et al. 2017; Tsurumaru et al. 2015). Furthermore, cluster analysis and studies on plant–microbial interactions and metabolism have been conducted in microorganisms that could not be previously cultured. However, these have mainly focused on rhizosphere bacteria, with studies on endophytic bacteria limited to rice (Sengupta et al. 2017), grape (Campisano et al. 2014), tomato (Tian et al. 2015), aloe (Akinsanya et al. 2015), peony (Yang et al. 2017), and other crops. Endophytic bacteria in Panax ginseng have been investigated by age, tissue, and region using culture-dependent methods (Hong et al. 2018; Khan Chowdhury et al. 2017; Park et al. 2012; Vendan et al. 2010), which has yielded only a partial understanding of the diversity, structure, and function of endophytic bacteria in P. ginseng.

Panax ginseng is frequently exposed to various stresses during the cultivation period as it is grown for a long time (minimum of 4 years) in the same place. Additionally, the rate of replant failure is high owing to large numbers of soil-borne pathogens and changes in soil microbial communities (Dong et al. 2016). Therefore, there is a need to elucidate the structure and function of the P. ginseng metagenome to develop measures to boost ginseng production.

Next-generation sequencing technology makes it possible to isolate bacterial genomes and exclude plant genome data from the whole genome dataset. Metagenome analysis of selected bacteria can be used to infer the diversity and functions of endophytic bacteria. In this study, we carried out the first metagenome analysis of endophytic bacteria of P. ginseng ‘Yunpoong’ by age (2–6 years). Our results provide new insights into the endophyte–host plant relationship, including previously unknown bacterial functions.

Materials and methods

Plant materials

We used 2–6-year-old ginseng root tissues of the ‘Yunpoong’ cultivar collected from the National Institute of Horticultural and Herbal Science of the Rural Development Administration in Chungbuk Province, Republic of Korea (127°45′13.14″E, 36°56′36.63″N). Root tissue samples were prepared from plants that were cultivated in the same field according to the ‘Ginseng GAP Standard Cultivation Guide’ developed by the Rural Development Administration (RDA, 2009)—which covers furrow directions, weed control, and disease/pest prevention—and were harvested between 2013 and 2018. A total of ten samples of each age were collected. To obtain bacterial endophytes, the soil on the root surface was washed with tap water and disinfected with 70% ethanol for 1 min and 12% sodium hypochlorite for 3 min, followed by fives rinses with sterile water. The successful removal of epiphytes by this disinfection method was confirmed by the absence of colonies in a culture of the final rinse water. The main root 1 cm from the rhizome and soil surface was used for analysis. Samples were frozen in liquid nitrogen and immediately ground using a TissueLyser (Qiagen, Hilden, Germany), and then stored at − 80 °C until use.

DNA extraction and shotgun metagenomic sequencing

Total genomic DNA was extracted from disinfected root tissue samples using the DNeasy Plant Mini Kit (Qiagen) according to manufacturer’s instructions. Each sample was sequenced according to the Illumina (San Diego, CA, USA) protocol. DNA quantity and quality were evaluated using PicoGreen and a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, 1 μg of genomic DNA was fragmented to obtain a 350-bp insert size using an ultrasonicator (Covaris, Woburn, MA, USA). The fragmented DNA was blunt-ended and phosphorylated followed by end repair, and an appropriate library size was selected using sample purification beads at different ratios. Additionally, ‘end repair’ was conducted using T4 DNA polymerase and T4 polynucleotide kinase. The 3′–5′ exonuclease activity of these enzymes removes 3′ overhangs and the polymerase activity fills in the 5′ overhangs. A single ‘A’ was added to the 3′ end, and Illumina adapters were ligated to the fragments. The final ligated product was analyzed by quantitative (q)PCR according to the qPCR Quantification Protocol Guide (Illumina Inc, San Diego, CA, USA), and library quality was evaluated using TapeStation DNA screentape D1000 (Agilent Technologies, Palo Alto CA, USA). Libraries were sequenced using the Illumina HiSeq X platform.

Analysis pipeline

Low-quality reads and adapter sequences were removed from the raw data via quality trimming using the Trimmomatic v 0.33 program (Bolger et al. 2014) with the minimum base quality, sliding window, average quality, and minimum read size specified. To extract unmapped reads, trimmed data were mapped to a reference genome using the BWA-mem program v 0.7.12 with the default option (Li and Durbin 2009). Mapped reads were counted using SAMtools v 1.2 (Li et al. 2009).

Metagenomic rapid annotation using subsystems technology (MG-RAST) analysis

The Panax unmapped reads for each plant age were uploaded to the MG-RAST server for gene prediction and annotation (Meyer et al. 2008). Taxonomic profiles were assigned to the SEED database with specific parameters (> 60% sequence identity to a subsystem and an e value cut-off of 10−5). Reference genome sequence data were obtained from the ginseng genome database (Jayakodi et al. 2018).

Results and discussion

Panax ginseng is usually cultivated for up to 4–5 years after planting a 1-year-old seedling (grown in a nursery for 1 year after sowing) in the field. Shotgun metagenomic analysis was carried out using 2–6-year-old root to obtain a profile of endophytic bacteria during the P. ginseng growing period in the field (Fig. 1). Trimmed reads were obtained from raw data for each sample by age. Unmapped data were obtained by mapping to the complete P. ginseng genome and were used to determine the type and functions of endophytic bacteria by MG-RAST analysis. The unmapped data were analyzed by identifying the sequence of microorganisms (while excluding the whole genome sequence of P. ginseng) from whole sequences of the analyzed sample. It was assumed that 3-year-old root samples (0.666%)—which had the highest unmapping rate in the complete ginseng genome—would have a relatively high diversity of microorganisms compared to 2-, 4-, 5-, and 6-year-old roots (Table 1). The rarefaction curve generated using MG-RAST confirmed that the 3-year-old root harbored the most species (Fig. 2).

Fig. 1.

Fig. 1

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Workflow for metagenomic analysis of ginseng plant

Table 1.

Statistical summary of Panax ginseng endophyte metagenomic data

Sample YP2 YP3 YP4 YP5 YP6
Total reads 373,376 694,368 209,679 186,431 273,228
Panax unmapping rate (%) 0.366 0.666 0.233 0.182 0.311
Sequence length (bp) 36,667,054 68,442,599 20,494,578 18,183,513 26,855,866
Mean sequence length (bp) 98 ± 9 99 ± 8 98 ± 10 98 ± 10 98 ± 9
Number of identified protein features 56,649 103,914 17,143 15,734 40,460
Number of identified rRNA features 235 275 142 118 348
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Fig. 2.

Fig. 2

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Rarefaction curves for obtained sequences of ginseng root-associated endophytes

Previous metagenomic studies have involved 16S rRNA gene sequencing and shotgun sequencing. However, 16S rRNA gene sequencing can be biased by unequal 16S rRNA gene amplification across species, while shotgun metagenomic sequencing does not yield reads of a sufficient length to detect the 16S rRNA gene of rare species in a sample (Shah et al. 2011). In the present study, we were able to obtain sufficient reads to classify bacterial species based on rarefaction curves generated with the shotgun metagenomic sequencing method (Fig. 2). This allowed us to remove the ginseng plant (host) DNA sequence from the obtained dataset, allowing a metagenomic analysis of the ginseng endophyte community.

For metagenomic analysis, MG-RAST was used to predict the structure and function of ginseng endophytic bacteria by plant age. A comparison of all unmapped sequences using the GenBank RefSeq database showed that approximately 81% of endophytes were bacteria (Fig. 3). To analyze only these microorganisms, we determined the distribution of endophytic bacteria in each bacterial sequence by age. Proteobacteria and Actinobacteria predominated at the phylum level, with the former being more abundant in 3–5-year-old root than in 2- and 6-year-old root (Fig. 4a). To investigate this in greater detail, we analyzed the distribution of endophytic bacteria at the class level. Alphaproteobacteria was abundant in 3–5-year-old root, whereas 2- and 6-year-old root showed similar profiles (Fig. 4b). A Venn diagram showed that 836 species were equally distributed across all ages, whereas 49 were specific to 3-year-old root, suggesting that plants at this age harbor a greater abundance of endophytic bacteria (Fig. 4c). Although many endophytic bacteria are not known to be beneficial to the host, they could potentially increase the frequency of mutualistic interactions. Among nine phyla—Actinobacteria, Bacteroidetes, Chlamydiae, Chloroflexi, Firmicutes, Fusobacteria, Proteobacteria, Spirochaetes, and Tenericutes—only three phyla, Actinobacteria, Chlamydiae, and Chloroflexi, were specific to 3-year-old root. Species found only in 3-year-old root, Streptomyces cyaneus and Dehalococcoides spp., are known to exhibit antifungal activities against six fungal pathogens and functions in bioremediation, respectively (Kunova et al. 2016; Tas et al. 2010). Additionally, the flavobacterium TRM1, which was identified by metagenomic analysis of the tomato rhizosphere, is successfully cultivated in vitro and is reported to inhibit the phytopathogen Ralstonia solanacearum in tomato (Kwak et al. 2018). This indicates that interactions between host and endophytes occur and that endophytes can contribute to host disease resistance.

Fig. 3.

Fig. 3

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Distribution of organisms detected in unmapped reads

Fig. 4.

Fig. 4

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Diversity of bacterial endophytes from 2–6-year-old ginseng root tissues. a, b Relative abundance at phylum (a) and genus (b) levels. c Venn diagram showing the number of specific and common species. d α-Diversity by MG-RAST analysis

The α diversity value obtained by MG-RAST analysis indicated that 3-year-old root had the highest diversity. The diversity was higher in 4- and 5-year-old root than in 2- and 6-year-old root, whereas their unmapped rates were lower (Fig. 4d). When endophytic bacteria of 4-year-old mountain-cultivated ginseng roots were identified by a culture-dependent method, the rank order of abundance at the phylum level was Gammaproteobacteria > Firmicutes > Betaproteobacteria > Actinobacteria (Khan Chowdhury et al. 2017); in 5-year-old ginseng root, the order was Firmicutes > Gammaproteobacteria > Actinobacteria (Cho et al. 2007). We previously analyzed the distribution of endophytic bacteria in the root of 1–6-year-old ginseng by 16S rRNA gene sequencing and found that the order of phylum-level abundance was Firmicutes > Actinobacteria > Gammaproteobacteria (Hong et al. 2018). The results of the present metagenomic analysis differ slightly from these previous reports: the rank order of abundance of endophytic bacteria in all samples (2–6-year-old ginseng root) was Alphaproteobacteria > Actinobacteria > Betaproteobacteria > Gammaproteobacteria. Endophytic bacteria inhabiting plants comprise mostly of Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes, with host-dependent differences in the predominant phylum (Hardoim et al. 2015). Indeed, 836 common species belonged to Proteobacteria and Firmicutes.

A comparison of previous classification results for endophytic bacteria of ginseng isolated by the culture-dependent method with those obtained by metagenomics analysis revealed differences in endophytic bacterial community profiles. This is expected since most bacteria in the natural world are viable but non-culturable. In addition, results obtained by the culture-independent method can differ from those of 16S rRNA gene sequencing and shotgun sequencing; in sugar beets, similar endophytic bacterial community compositions were detected by both methods but the relative abundance of taxa were differed (Tian et al. 2015).

We performed a SEED subsystem analysis to predict the function of endophytic bacteria by age. Although bacterial diversity varied with age, level 1 function did not differ significantly (Fig. 5a). This can be explained by the finding that bacteria assemble based on functional rather than taxonomic relatedness (Burke et al. 2011). Furthermore, it is reported that the function of endophytic bacteria does not depend on taxonomic classification but on host and environmental factors (Hardoim et al. 2015).

Fig. 5.

Fig. 5

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Functional categories by SEED subsystems. a, b Comparison of metagenomic profiles at SEED subsystem level 1 (a) or level 3 (b)

At the lower level, siderophore and auxin-related traits—which are known to promote plant growth—were more highly represented in 3-year-old root (Fig. 5b). This suggests that interactions between endophytic bacteria and ginseng plants at this growth stage are beneficial for root growth. Additionally, we used BLASTn to examine the representation of six genes involved in promoting plant growth identified in our obtained sequence data (Table 2). It is known that bacteria containing the enzyme 1-aminocyclopropane-1-carboxylic acid deaminase promote growth of host by lowering plant ethylene levels (Bernard 2014). In addition, inorganic phosphate solubilization and assimilation of nitrogen are key mechanisms of plant growth promotion (Crawford 1995; Oteino et al. 2015). Several endophytic bacteria solubilize the phosphate complexes and convert them into ortho-phosphate that is available for plant up-take and utilization (Oteino et al. 2015). All six genes were detected in 3-year-old P. ginseng and the remaining samples expressed one or two of the genes, confirming that P. ginseng is actively growing in the third year. In fact, P. ginseng roots increase in length until the second year, after which their lateral branches divide and increase in volume (Park et al. 2013). Thus, the unique growth pattern of ginseng may be directly related to the distribution of endophytic bacteria.

Table 2.

Frequency of putative functional genes in the Panax ginseng bacterial community detected by BLASTn

Gene ID of UniRef Length (bp) Frequency per 105 reads
YP2 YP3 YP4 YP5 YP6
ACC deaminase A5EJ46 1020 ND 0.60 ND ND ND
Phosphate solubilization
 4-phytase B0UQX3 1635 ND 0.37 ND ND ND
Methanol utilization
 XoxF C5ATJ3 1800 0.59 0.34 ND ND 0.85
N metabolism-related genes
 glnA A0R079 1473 ND 0.43 2.49 ND 5.34
 gltB Q05755 4545 0.47 0.27 0.79 0.85 ND
 nirB A6UI45 2463 ND 0.25 ND ND ND
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ND not detected

The 6-year-old P. ginseng root showed the lowest diversity in this study. It is supposed that low microbial diversity is due to the effects of chemical pesticides that are continuously applied to the plant over a long period (Khan Chowdhury et al. 2017). A study in bellflower and grape revealed marked differences in their endophytic bacterial communities and functions (Andreolli et al. 2016; Asraful Islam et al. 2010) that may be attributable to fluctuation or reshaping of the bacterial community under different conditions (e.g., plant growth stage or surrounding environment) (Fuchs et al. 2017; Mocali et al. 2003; Wagner et al. 2016).

Concluding remarks

We performed a metagenome analysis to clarify the structure and function of endophytic bacterial communities in 2–6-year-old ginseng. The results demonstrate that endophytic bacterial profiles of ginseng root vary according to age, with 3-year-old plants having the greatest diversity of endophytic bacteria. Our observation that traits related to plant growth were most highly represented in 3-year-old ginseng suggests that the plant growth mechanism can be explained at least in part by plant–microbe interaction. Our findings provide a basis for potentially improving P. ginseng cultivation by exploiting the beneficial properties of endophytic bacteria.

Acknowledgements

This work was supported by the Cooperative Research Program for Agriculture Science & Technology Development (Project no. PJ 01312412019), Rural Development Administration, Republic of Korea; and by the 2019 Postdoctoral Fellowship Program of National Institute of Horticultural and Herbal Science, Rural Development Administration, Republic of Korea.

Data accessibility

This BioProject has been deposited in NCBI under accession number PRJNA530282. Sequences obtained in this work have been deposited in the NCBI Sequence Read Archive under accession number SRP190953.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

This BioProject has been deposited in NCBI under accession number PRJNA530282. Sequences obtained in this work have been deposited in the NCBI Sequence Read Archive under accession number SRP190953.

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