Diversity and Composition of Bacterial Endophytes Among Plant Parts Of
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Dong et al. Chin Med (2018) 13:41 https://doi.org/10.1186/s13020-018-0198-5 Chinese Medicine RESEARCH Open Access Diversity and composition of bacterial endophytes among plant parts of Panax notoginseng Linlin Dong1, Ruiyang Cheng1, Lina Xiao1, Fugang Wei2, Guangfei Wei1, Jiang Xu1, Yong Wang3, Xiaotong Guo4, Zhongjian Chen3 and Shilin Chen1* Abstract Background: Bacterial endophytes are widespread inhabitants inside plant tissues that play crucial roles in plant growth and biotransformation. This study aimed to ofer information for the exploitation of endophytes by analyzing the bacterial endophytes in diferent parts of Panax notoginseng. Methods: We used high-throughput sequencing methods to analyze the diversity and composition of bacterial endophytes from diferent parts of P. notoginseng. Results: A total of 174,761 classifed sequences were obtained from the analysis of 16S ribosomal RNA in diferent parts of P. notoginseng. Its fbril displayed the highest diversity of bacterial endophytes. Principal coordinate analysis revealed that the compositions of the bacterial endophytes from aboveground parts (fower, leaf, and stem) difered from that of underground parts (root and fbril). The abundances of Conexibacter, Gemmatimonas, Holophaga, Luteoli- bacter, Methylophilus, Prosthecobacter, and Solirubrobacter were signifcantly higher in the aboveground parts than in the underground parts, whereas the abundances of Bradyrhizobium, Novosphingobium, Phenylobacterium, Sphingo- bium, and Steroidobacter were markedly lower in the aboveground parts. Conclusions: Our results elucidated the comprehensive diversity and composition profles of bacterial endophytes in diferent parts of 3-year-old P. notoginseng. Our data ofered pivotal information to clarify the role of endophytes in the production of P. notoginseng and its important metabolites. Keywords: Panax notoginseng, Endophytes, Plant parts, High-throughput sequencing, 16S ribosomal RNA Background even seedling death [6, 7]. Approximately 8–10 years of Panax notoginseng is renowned for its remarkable anti- crop rotation are necessary for improving the soil con- hypertensive, antithrombotic, anti-atherosclerotic, and ditions for planted P. notoginseng [8]. P. notoginseng has neuroprotective bioactivities, making it one of the most a narrow ecological range, and its production mainly valuable ingredients in staple household medicines [1–3]. occurs in Wenshan, Yunnan Province, where the climatic Protopanaxadiol and protopanaxatriol saponins are the and soil conditions are optimal for its cultivation. Never- main active compounds detected in the diferent parts theless, arable soils available for P. notoginseng cultivation of P. notoginseng [4, 5]. P. notoginseng is a perennial plant are becoming scarce. cultivated in fxed plots, and continuous cropping leads Endophytic bacteria localized inside plant tissues to decreased productivity, reduced tuber quality, and have shown no negative efect on their host plants [9]. Bacteria inhabit diferent plant tissues, including rhizo- sphere, root, leaf, and stem [10]. Bacterial endophytes *Correspondence: [email protected] play key roles in improving plant growth, increasing 1 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China tolerance against biotic factors, and producing sec- Full list of author information is available at the end of the article ondary metabolites [11, 12]. Song et al. reported that © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dong et al. Chin Med (2018) 13:41 Page 2 of 9 endophytic Bacillus altitudinis isolated from Panax Methods ginseng enhanced ginsenoside accumulation [13]. Gao Processing of samples et al. reported that Paenibacillus polymyxa isolated Tree-year-old P. notoginseng plants were collected from from P. ginseng leaves improved plant growth, increased Wenshan, Yunnan Province, China, which is the main ginsenoside concentration, and reduced morbidity [14]. production area of P. notoginseng. Tese plant samples Endophytes stimulated secondary metabolites and were used to analyze the bacterial endophytes in diferent enhanced plant growth. Numerous works highlighted parts of P. notoginseng. Six plants were randomly gath- that the composition of the bacterial endophytes were ered from one plantation and served as one sample in our infuenced by plant species, parts, and growth stage test sites of Wenshan Miaoxiang Notoginseng Technol- [11, 15]. Analysis of the diversity and composition ogy, Co., Ltd. in August. Tere were three replicates from of endophytes in plant parts could provide valuable three plantations. Te fowers (Fl), leaves (Le), stems (St), resources for plant growth promotion and biotransfor- roots (Ro), and fbers (Fi) of all samples were separated, mation [16]. Although the diversity and composition of washed with running tap water, and rinsed thrice with root endogenous bacteria in P. notoginseng have been distilled water. A single sample consisted of 1 g of each described [17], limited information is available on the part from six plants as one sample. To sterilize the sur- endophytic community in diferent parts of P. notogin- face of the plant parts, the samples from each part were seng. Tus, the diversity and composition of bacterial successively immersed in 70% ethanol for 5 min, 2.5% endophytes must be investigated to exploit the agro- sodium hypochlorite for 1–2 min, and 70% ethanol for nomical and metabolic potential of P. notoginseng. 1 min, and then rinsed fve times with sterile Millipore Cultivation-independent methods can facilitate a water. Te last portion of the washing water was inocu- rapid analysis of vast samples and provide reliable lated in Luria–Bertani agar at 37 °C for 24 h to validate information on the diversity and composition of endo- sterilization efciency. A total of 15 samples were stored phytic bacteria [18]. Many studies have explored rhizo- at − 80 °C until DNA extraction. sphere and plant-associated bacterial communities by using high-throughput sequencing analysis [19–21]. DNA extraction, polymerase chain reaction (PCR) Metagenetic methods for analyzing endophyte com- amplifcation, and sequence processing munities would provide deeper insight into the diver- Te total genomic DNA was extracted from all plant sity and composition of bacterial endophytes, thereby parts by using the MOBIO PowerSoil ® Kit (MOBIO Lab- leading to the potential discovery of new endophytes oratories, Inc., Carlsbad, CA, USA) in accordance with [22, 23]. Checcucci et al. reported the high taxo- the manufacturer’s instructions. Te DNA quality of each nomic diversity of bacterial endophytes in the leaves sample was confrmed by utilizing a NanoDrop spectro- of Tymus spp. by using 16S rRNA gene metagenomic photometer (Termo Fisher Scientifc, Model 2000, MA, sequencing [24]. A total of 29 culturable bacterial endo- USA) and stored at − 20 °C for further PCR amplifcation. phytes have been identifed in the tissues of Aloe vera Bacterial 16S rRNA V1 hypervariable region genes were and characterized to 13 genera [25]. Nevertheless, cul- amplifed by using the universal primers 27F/338R [29]. ture-dependent biodiversity studies on endophytic bac- Te forward and reverse primers contained an 8 bp bar- terial communities remain scarce [19]. Pyrosequencing code (Additional fle 1: Table S1). PCRs were performed could detect low-abundance bacteria in leaf salad vege- as described by Dong et al. with slight modifcations [21]. tables that could not be identifed by culture-dependent Te reaction systems were denatured at 94 °C for 3 min methods [26]. Additional, high-throughput sequencing and then amplifed for 25 cycles at 94 °C for 45 s, 55 °C also used in the analysis of soil microbial communi- for 30 s, and 72 °C for 60 s. A fnal extension of 10 min ties, and this method efectively revealed the changes was added at the end of the program. Negative controls in diversity of soil microbial communities in soils dur- (no templates) were included to check DNA contami- ing the cultivation of Panax plants [21, 27, 28]. In the nation of the primer or the sample. PCR products from present study, high-throughput sequencing analysis of each sample were separated with 1% agarose gel, purifed 16S ribosomal RNA (rRNA) genes was conducted to with a MinElute Gel Extraction Kit (Qiagen, Valencia, describe the diversity and composition of associations CA, USA), and quantifed with a Quant-iT PicoGreen among diferent parts of P. notoginseng. Te results dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). Te clarifed the tissue-wise diversity of bacterial endo- amplicons were pooled in equimolar ratios. Te amplicon phytes in the samples collected from P. notoginseng as libraries were paired-end sequenced (2 × 250) by using an well as expanded the knowledge on plant–microbe rela- Illumina MiSeq platform in accordance with the manu- tionships and the potential properties for plant growth facturer’s protocol. promotion and biotransformation.