Background
Panax notoginseng is renowned for its remarkable antihypertensive, antithrombotic, anti-atherosclerotic, and neuroprotective bioactivities, making it one of the most valuable ingredients in staple household medicines [
1‐
3]. Protopanaxadiol and protopanaxatriol saponins are the main active compounds detected in the different parts of
P. notoginseng [
4,
5].
P. notoginseng is a perennial plant cultivated in fixed plots, and continuous cropping leads to decreased productivity, reduced tuber quality, and even seedling death [
6,
7]. Approximately 8–10 years of crop rotation are necessary for improving the soil conditions for planted
P. notoginseng [
8].
P. notoginseng has a narrow ecological range, and its production mainly occurs in Wenshan, Yunnan Province, where the climatic and soil conditions are optimal for its cultivation. Nevertheless, arable soils available for
P. notoginseng cultivation are becoming scarce.
Endophytic bacteria localized inside plant tissues have shown no negative effect on their host plants [
9]. Bacteria inhabit different plant tissues, including rhizosphere, root, leaf, and stem [
10]. Bacterial endophytes play key roles in improving plant growth, increasing tolerance against biotic factors, and producing secondary metabolites [
11,
12]. Song et al. reported that endophytic
Bacillus altitudinis isolated from
Panax ginseng enhanced ginsenoside accumulation [
13]. Gao et al. reported that
Paenibacillus polymyxa isolated from
P. ginseng leaves improved plant growth, increased ginsenoside concentration, and reduced morbidity [
14]. Endophytes stimulated secondary metabolites and enhanced plant growth. Numerous works highlighted that the composition of the bacterial endophytes were influenced by plant species, parts, and growth stage [
11,
15]. Analysis of the diversity and composition of endophytes in plant parts could provide valuable resources for plant growth promotion and biotransformation [
16]. Although the diversity and composition of root endogenous bacteria in
P. notoginseng have been described [
17], limited information is available on the endophytic community in different parts of
P. notoginseng. Thus, the diversity and composition of bacterial endophytes must be investigated to exploit the agronomical and metabolic potential of
P. notoginseng.
Cultivation-independent methods can facilitate a rapid analysis of vast samples and provide reliable information on the diversity and composition of endophytic bacteria [
18]. Many studies have explored rhizosphere and plant-associated bacterial communities by using high-throughput sequencing analysis [
19‐
21]. Metagenetic methods for analyzing endophyte communities would provide deeper insight into the diversity and composition of bacterial endophytes, thereby leading to the potential discovery of new endophytes [
22,
23]. Checcucci et al. reported the high taxonomic diversity of bacterial endophytes in the leaves of
Thymus spp. by using 16S rRNA gene metagenomic sequencing [
24]. A total of 29 culturable bacterial endophytes have been identified in the tissues of
Aloe vera and characterized to 13 genera [
25]. Nevertheless, culture-dependent biodiversity studies on endophytic bacterial communities remain scarce [
19]. Pyrosequencing could detect low-abundance bacteria in leaf salad vegetables that could not be identified by culture-dependent methods [
26]. Additional, high-throughput sequencing also used in the analysis of soil microbial communities, and this method effectively revealed the changes in diversity of soil microbial communities in soils during the cultivation of
Panax plants [
21,
27,
28]. In the present study, high-throughput sequencing analysis of 16S ribosomal RNA (rRNA) genes was conducted to describe the diversity and composition of associations among different parts of
P. notoginseng. The results clarified the tissue-wise diversity of bacterial endophytes in the samples collected from
P. notoginseng as well as expanded the knowledge on plant–microbe relationships and the potential properties for plant growth promotion and biotransformation.
Methods
Processing of samples
Three-year-old P. notoginseng plants were collected from Wenshan, Yunnan Province, China, which is the main production area of P. notoginseng. These plant samples were used to analyze the bacterial endophytes in different parts of P. notoginseng. Six plants were randomly gathered from one plantation and served as one sample in our test sites of Wenshan Miaoxiang Notoginseng Technology, Co., Ltd. in August. There were three replicates from three plantations. The flowers (Fl), leaves (Le), stems (St), roots (Ro), and fibers (Fi) of all samples were separated, washed with running tap water, and rinsed thrice with distilled water. A single sample consisted of 1 g of each part from six plants as one sample. To sterilize the surface of the plant parts, the samples from each part were successively immersed in 70% ethanol for 5 min, 2.5% sodium hypochlorite for 1–2 min, and 70% ethanol for 1 min, and then rinsed five times with sterile Millipore water. The last portion of the washing water was inoculated in Luria–Bertani agar at 37 °C for 24 h to validate sterilization efficiency. A total of 15 samples were stored at − 80 °C until DNA extraction.
The total genomic DNA was extracted from all plant parts by using the MOBIO PowerSoil
® Kit (MOBIO Laboratories, Inc., Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. The DNA quality of each sample was confirmed by utilizing a NanoDrop spectrophotometer (Thermo Fisher Scientific, Model 2000, MA, USA) and stored at − 20 °C for further PCR amplification. Bacterial 16S rRNA V1 hypervariable region genes were amplified by using the universal primers 27F/338R [
29]. The forward and reverse primers contained an 8 bp barcode (Additional file
1: Table S1). PCRs were performed as described by Dong et al. with slight modifications [
21]. The reaction systems were denatured at 94 °C for 3 min and then amplified for 25 cycles at 94 °C for 45 s, 55 °C for 30 s, and 72 °C for 60 s. A final extension of 10 min was added at the end of the program. Negative controls (no templates) were included to check DNA contamination of the primer or the sample. PCR products from each sample were separated with 1% agarose gel, purified with a MinElute Gel Extraction Kit (Qiagen, Valencia, CA, USA), and quantified with a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The amplicons were pooled in equimolar ratios. The amplicon libraries were paired-end sequenced (2 × 250) by using an Illumina MiSeq platform in accordance with the manufacturer’s protocol.
Data analysis
The data were processed by utilizing the QIIME pipeline [
30]. Bacterial sequences were trimmed and assigned to each sample based on their barcodes. Sequences were binned into operational taxonomic units (OTUs) at 97% similarity level by using UPARSE (version 7.1
http://drive5.com/uparse/). Chimeric sequences were identified and removed by using UCHIME. The phylogenetic affiliation of each 16S rRNA gene sequence was analyzed by using a RDP Classifier (
http://rdp.cme.msu.edu/) against the Silva (SSU123) 16S rRNA database at a confidence threshold of 70% [
31]. Rarefaction analysis based on Mothur v.1.21.1 was conducted to reveal the diversity indices, including Chao 1 and Shannon [
32]. Principal coordinate analysis (PCoA) was performed to examine dissimilarities in the community composition among samples on the basis of Bray–Curtis distance metrics [
33]. Statistical analyses were performed by using the
R package [
34].
Statistical analyses
SPSS version 16.0 was used for the statistical analyses (SPSS Inc., Chicago, IL, USA). The data were presented as mean ± SD of n = 3. No adjustments were implemented for multiple comparisons. The parameters were obtained for all treatment replicates and subjected one-way ANOVA.
The Minimum Standards of Reporting Checklist contains details of the details of the experimental design, and statistics, and resources used in this study (Additional file
2).
Discussion
In this study, the diversity of bacterial endophytes was associated with the different parts of
P. notoginseng. Chao 1 and
H’ indices indicated that the fibril samples had the highest diversity among all of the samples from the different parts. Chao 1 revealed that
Stellera chamaejasme L. displayed an increasing trend in species richness from the root samples to the stem and leaf samples [
19]. The OTUs of the bacterial endophytes were randomly distributed among plant species and organs, and Chao 1 also revealed that the diversity of
Santiria apiculate and
Rothmannia macrophylla in the root samples was higher than that in the leaf samples [
35]. PCoA showed that samples from the aboveground parts were distinguishable from those from the underground parts. Principal component analysis (PCA) revealed that the leaf and stem samples of
S. chamaejasme L. were clustered together and were different from the plots for the root [
19], and that the stem and leaf samples of poplar trees were distinguishable from the root samples [
10]. The bacterial endophytes from the fibril had the highest diversity.
In this study, Proteobacteria, Actinobacteria, Verrucomicrobia, Bacteroidetes, Acidobacteria, and Firmicutes were the main bacterial communities in
P. notoginseng plants. A previous study detected Proteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria in the
P. notoginseng root [
17]. Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes were found in
P. ginseng roots by using a culture-dependent method [
16]. More than 300 endophytic actinobacteria and bacteria belonging to
Rhodococcus,
Brevibacterium,
Nocardioides,
Streptomyces,
Microbacterium,
Nocardiopsis,
Brachybacterium,
Tsukamurella,
Arthrobacter, and
Pseudonocardia were isolated from different tissues of
Dracaena cochinchinensis L. [
12]. The plant species influenced the selection of endophytes. The plant parts of
P. notoginseng represented the ecological niches for bacterial endophytes.
The composition of bacterial endophytes from the aboveground parts varied from that of the underground parts. The composition of bacterial endophytes was associated with the plant compartments [
10]. The relative abundances of the bacterial endophytes in all samples showed considerable variations at the phylum and genus levels [
19]. The relative abundances of the bacterial endophytes, including
Conexibacter,
Gemmatimonas,
Holophaga,
Luteolibacter,
Methylophilus,
Prosthecobacter,
Solirubrobacter,
Bradyrhizobium,
Novosphingobium,
Phenylobacterium,
Sphingobium, and
Steroidobacter, in the aboveground and underground parts differed significantly. Evident strains are
Gemmatimonas, Bradyrhizobium,
Novosphingobium, and
Sphingobium, which can solubilize insoluble elements, induce plant stress resistance or produce antifungal antibiotics [
36‐
39]. Endophytic
Bacillus altitudinis served as elicitors of biomass and ginsenoside production [
13]. Bacterial endophytes from
Zea displayed anti-fungal activity against two fungal pathogens [
40]. Li et al. have reported that the domain genera included
Rhizobium,
Sulfurospirillum,
Uliginosibacterium,
Pseudomonas,
Aeromonas and
Bacteroides, all of which could fix nitrogen and improve plant growth [
41]. The fungal endophytes communities in
Monarda citriodora expressed anticancer and antimicrobial activities [
42]. In view of the roles played by endophytes in plant growth and biotransformation, our findings contribute to the expansion of endophyte use in the production of
P. notoginseng and its important metabolites. The information on the differences of endophytes in the aboveground and underground parts can serve as basis for the selection of functional bacteria. Importantly, higher saponins contents were detected in harvest 3-year-old
P. notoginseng plants [
43,
44]. Endophytes increased ginsenoside concentration and reduced morbidity [
13,
14]. Thus, 3-year-old
P. notoginseng plants served as the proper samples to analyze the endophytes. Additionally, the diversity of bacterial endophytes showed richness than fungal endophytes or exogenous bacteria in hour study (data not shown), and we focused on bacterial endophytes in different parts of
P. notoginseng.
Authors’ contributions
LD designed the work, analyzed the data, and wrote the manuscript. RC analyzed the data and collected samples. LX, GW and FW performed the experiment. JX and XG analyzed the data. YW and ZC performed the field experiment. SC designed the work and wrote this manuscript. All authors read and approved the final manuscript.