Community Analysis of Endophytic Bacteria from the Seeds of the Medicinal Plant Panax notoginseng

Panax notoginseng is a traditional Chinese medicine. The roots of P. notoginseng can be used for treatment of diseases and raw materials in Chinese medicinal products. High yield and quality roots require cultivation in shade and humid conditions for 3 years. The long period cultivation makes P. notoginseng vulnerable to infect by pathogens. So control diseases are vital for the high yield and quality of P. notoginseng. The seed is the carrier systems of many probiotics and pathogens. To explore the indigenous bacterial community diversity, the endophytic bacteria from the seeds of the medicinal plant P. notoginseng were isolated and identified using traditional cultivation methods in combination with molecular technique. A total of 137 endophytic bacteria strains were isolated. The 16S rDNA of these strains was amplified and subjected to amplified ribosomal DNA restriction analysis (ARDRA) with restriction enzyme HaeIII. All the isolated strains were grouped into 9 OTUs (Operational Taxonomic Units) on the basis of the similarity of the ARDRA band profiles. Each representative strain of 9 OTUs was selected for sequencing. γ-proteobacteria was the most dominant group among the isolates (98.5%), containing eight genera. Pseudomonas was the most dominant genus (58 of 135 isolates), whose isolates occurred in the seeds collected from all three places. The second dominant genus was Enterobacter (20.7%), followed by uncultured bacterium (14.8%) and Stenotrophomonas (10.4%). Among the six areas sampled, endophytic bacteria in the seeds collected from Panlong of Yanshan exhibited species diversity and contained the most isolates. These results suggest an abundant diversity of bacterial community within the seeds of P. notoginseng. These data provide insights into monitoring the seed health and disease outbreak during seeding.


Introduction
Endophytic bacteria ubiquitously inhabit a majority of plant species (Lodewyck et al., 2002).These organisms can be isolated from surface-disinfected plant tissues, including seeds, roots, stems and leaves and are not harmful to the hosts (Hallmann et al., 1998).Endophytic bacteria may promote plant growth and suppress plant diseases (Feng et al., 2006).Several studies have reviewed endophytic bacteria community structures and their potential biological functions (Senthilkumar et al., 2011;Sturz et al., 2000).
Panax notoginseng is a traditional Chinese medicine plant specifically grown in the Wenshan region of Yunnan Province.The root of P. notoginseng is often used to treat cardiovascular diseases, inflammation, different body pains, trauma, and internal and external bleeding due to injury (Sun et al., 2005).P. notoginseng is a 3-year-old plant.High quality roots require 3-year cultivation in the shade and humid conditions.The long period growth makes P. notoginseng vulnerable to be infected by pathogens.The root-rot disease is the most destructive one (Sun et al., 2004), which was caused by soil-borne fungal pathogens (including Alternaria panax, Alternaria tenuis, Cylindrocarpon destructans, Cylindrocarpon didynum, Fusarium solani, Fusarium oxysporum, Phytophthora cactorum, Phoma herbarum and Rhizoctonia solani), bacterial pathogens (Pseudomonas sp. and Ralstonia sp.), and parasitic nematodes (such as Ditylenchus sp., Rhabditis elegans and Meloidogyne spp.) (Miao et al., 2006).Control diseases are vital for the high yield and quality of P. notoginseng.
The seeds of plants are the carrier systems of many probiotics and pathogens that play an important role in formation of rhizosphere microbial communities (Patkowska, 2001).However, studies concerning the interaction of the microbial community and plant seed genotype are lacking.Therefore, a better study of endophytic bacteria may help increase the current understanding of their function and potential role for the development of a more sustainable system for crop production.Few studies concerning endophytic bacterial community structures and their biological functions in Panax spp.have been reported, with the exception of reports on the endophytic bacterial community (Cho et al., 2007;Vendan et al., 2010).Ma et al. (2013) isolated 1000 endophytic bacterial strains from the root, stems, petioles, leaves and seeds of P. notoginseng, of which 104 strains exhibited antagonistic properties against at least one of three major pathogens (F.oxysporum, Ralstonia sp. and Meloidogyne hapla) related to the root-rot disease of P. notoginseng.Therefore, knowledge concerning the endophytic bacterial structures and species is vital for the growth of Panax plants, the monitoring of seed health and the control of seedling disease.Thus, the aims of the present study were to characterize the endophytic bacteria present in the seeds of the medicinal plant P. notoginseng, to obtain a better understanding of endophytic bacterial community structures and diversities and to identify potential biological control candidates against pathogens leading to seedling and root diseases.

Microorganisms and Culture Conditions
The bacterial strains were cultured on Luria-Bertani (LB) solid medium (peptone 10 g, yeast extract 5 g, NaCl 10 g, agar powder 15 g, and distilled water 1000 mL, pH 7.2) or in LB liquid broth at 37 °C.

Isolation and Purification of Endophytic Bacteria
The seeds were collected from 1-3-year-old healthy P. notoginseng plants, cultivated in Yanshan County, Maguan County, Guangnan County, Xichou County, Wenshan County and Qiubei County of the Wenshan region, Yunnan Province, China.
The samples were disinfected according to Li et al. (2010), with some modifications.The seeds were rinsed three times with sterile water, and subsequently, the moisture was absorbed using filter paper.The seeds were sterilized with 70% ethanol for 3 min, immersed in 2.6% sodium hypochlorite solution for 5 min, soaked in 70% ethanol for 30 s, and subsequently washed three times with sterile water.The water samples of the last rinse were inoculated onto LB agar plates as a negative control.Simultaneously, the surface sterilized seeds were pressed onto an LB agar plate to test the sterilization efficiency.The seeds that were not detected as contaminated by cultivable microorganisms were considered successfully surface disinfected and were subsequently used for the isolation of endophytic bacteria (Schulz et al., 1993).
The surface sterilized seeds were divided into three groups.Five seeds in each group were homogenized with 2 mL of sterile water.The homogenates were diluted 1000-fold, and 100-µL dilutions were then spread onto LB agar plates.Each sample was replicated three times.The plates were cultured at 37 °C for 72 h.The bacterial colonies with obvious morphological differences were purified.Colonies without distinct morphological differences were randomly selected.

DNA Extraction from Bacteria
The purified strains were inoculated in LB liquid medium and shaken at 200 rpm for 12 h at 37 °C.The cultures were centrifuged and subsequently collected.Genomic DNA was extracted using the Genomic DNA Purification Kit (TIANGEN Biotech, Beijing, China) according to the manufacturer's instructions.

Amplification of the Bacterial 16S rRNA Gene
The genomic DNA was used as the PCR template.A pair of primers, 799f and 1492r [13], were used to amplify the 16S rDNA.In total, a 20-µL PCR reaction mixture contained 1 µL of genomic DNA, 1 µL of each primer (10 µM), and 1 µL 2 × Es Taq MasterMix (Es Taq DNA Polymerase, 2 × Es Taq PCR buffer, 3 mM MgCl 2 , and 400 µM dNTP) (ComWin Biotech, Beijing, China).After initial denaturing at 94 °C for 3 min, thermal cycling proceeded with, denaturing at 94 °C for 30 s, annealing at 50 °C for 30 s, and elongation at 72 °C for 45 s.At the end of 30 cycles, a final extension step was performed at 72 °C for 10 min.jas.ccsenet.

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A single c DNA extra isolates.G (Figure 1   Endophytes have been considered as abundant sources for probiotics.Ratnaweera et al. (2013) isolated eight endophytic fungi from Opuntia dillenii, seven of which exhibited antimicrobial activity.The most biologically active species is Fusarium sp., and the second most active is Aspergillus niger.Chadha et al. (2015) suggested that plant endophytic fungi can protect plants against various pathogen and pests and help plants survive under harsh biological or abiotic stresses.Tantirungkij et al. (2015) reported that rice leaves harbor several new yeast strains.Khan et al. (2015) isolated two fungal strains, Fusarium tricinctum RSF-4L and Alternaria alternata RSF-6L, which promote the growth of the host by phytohormone secretion.Ma et al. (2013) isolated endophytic bacteria from P. notoginseng, and 104 strains showed antagonism against Fusarium oxysporum, Ralstonia sp. and Meloidogyne hapla, which are three major pathogens associated with the root-rot disease complex of P. notoginseng.Shahzad et al. (2016) isolated an endophytic Bacillus amyloliquefaciens with the potential to produce gibberellins (GAs) and that plays a role in improving host-plant physiology.
In the present study, the bacterial endophytes isolated from the seeds of P. notoginseng belonged to four bacterial groups, including γ-proteobacteria and Firmicutes, Enterobacter, uncultured bacteria and Firmicutes.These results suggest that P. notoginseng seeds carry abundant microbial resources.Among the isolated bacteria, the dominant bacteria was Pseudomonas sp., followed by Enterobacter sp., uncultured bacteria clones, and Stenotrophomonas sp.To our knowledge, this report is the first comprehensive study on the isolation of endophytic bacteria from P. notoginseng seeds.
Strains isolated from the seeds may have biological activity against pathogens or perhaps are pathogens leading to diseases that occur during seed germination, seedling growth or plant development.Some Pseudomonas strains serve as plant growth-promoting rhizobacteria (PGPR) or biological control agents against plant pathogens (de Bruijn et al., 2007;Raaijmakers et al. 2010).Pseudomonas sp. is also one of bacterial pathogens that cause P. notoginseng root-rot disease (Miao et al., 2006).In the present study, the dominant endophytic bacteria were Pseudomonas sp.Whether the subsequent harboring of these Pseudomonas sp.strains leads to disease or plays a beneficial role on plants requires further research.Stenotrophomonas maltophilia has been reported to promote plant growth due to its production of phytohormones (Park et al., 2005;Naz et al., 2009) and to be a biological control agent due to its production of antibacterial compounds and secretion of fungicidal metabolites (Messiha et al., 2007;Taghavi et al., 2009).Pectobacterium carotovorum is a plant pathogen with a diverse host range and that causes bacterial soft rot (Mansfield et al., 2012).Enterobacter aerogenes is a nosocomial and pathogenic bacterium that causes opportunistic human infections.Whether these pathogens cause diseases on P. notoginseng remains unknown.Therefore, the isolation of these endophytic bacterial strains not only helps to further current understanding of the outbreak of seedling or plant diseases, but also provides good candidates for biological control against soil-borne root diseases.

Table 2 .
Similarity of the16S rDNA sequences of partial endophytic bacterial strains from P. notoginseng seeds