Endophytic Actinobacteria Associated with Dracaena cochinchinensis Lour.: Isolation, Diversity, and Their Cytotoxic Activities

Dracaena cochinchinensis Lour. is an ethnomedicinally important plant used in traditional Chinese medicine known as dragon's blood. Excessive utilization of the plant for extraction of dragon's blood had resulted in the destruction of the important niche. During a study to provide a sustainable way of utilizing the resources, the endophytic Actinobacteria associated with the plant were explored for potential utilization of their medicinal properties. Three hundred and four endophytic Actinobacteria belonging to the genera Streptomyces, Nocardiopsis, Brevibacterium, Microbacterium, Tsukamurella, Arthrobacter, Brachybacterium, Nocardia, Rhodococcus, Kocuria, Nocardioides, and Pseudonocardia were isolated from different tissues of D. cochinchinensis Lour. Of these, 17 strains having antimicrobial and anthracyclines-producing activities were further selected for screening of antifungal and cytotoxic activities against two human cancer cell lines, MCF-7 and Hep G2. Ten of these selected endophytic Actinobacteria showed antifungal activities against at least one of the fungal pathogens, of which three strains exhibited cytotoxic activities with IC50-values ranging between 3 and 33 μg·mL−1. Frequencies for the presence of biosynthetic genes, polyketide synthase- (PKS-) I, PKS-II, and nonribosomal peptide synthetase (NRPS) among these 17 selected bioactive Actinobacteria were 29.4%, 70.6%, and 23.5%, respectively. The results indicated that the medicinal plant D. cochinchinensis Lour. is a good niche of biologically important metabolites-producing Actinobacteria.


Introduction
Actinobacteria, especially the genus Streptomyces, are major producers of bioactive metabolites [1] and account for nearly 75% of the total antibiotic production available commercially [2,3]. A few decades ago, antibiotics were considered as wonder drugs since they warded off deadly pathogens leading to eradication of infectious diseases. However, the unprecedented deployment of antibiotics over a period of time has resulted in evolution of multidrug-resistant pathogens. There is increasing attention to bioprospecting of Actinobacteria from different biotopes. With limiting bioresources, it is now imperative for search of unexplored or underexplored habitats. One such overlooked and promising niche is the 2 BioMed Research International inner tissues of plants, especially those with ethnomedicinal value [4][5][6][7][8][9][10].
The plant Dracaena cochinchinensis Lour. has been used as a traditional folk medicine in the oriental countries including China [11]. D. cochinchinensis Lour. has many medicinally important properties, like antimicrobial, antiviral, antitumor, cytotoxic, analgesic, antioxidant, anti-inflammatory, haemostatic, antidiuretic, antiulcer, and wound healing activities [10,12]. The plant is the source of deep red resin having medicinal properties which is also known as dragon's blood. The main components of dragon's blood are flavonoids and stilbenoids [13]. Apart from its medicinal use, it also finds applications as colouring materials and wood varnish [12]. The slow growth of the plant along with low yield of dragon's blood extracts, however, led to the destruction of large number of these plants, thereby endangering the plant. The current study described the diversity of culturable Actinobacteria associated with this medicinal plant and also indicated the cytotoxic potential of these Actinobacteria. The study, in a way, proposed a means for sustainable use of the plant resources without destroying the natural niche. The plant samples were packed in sterile plastics, taken to the laboratory, and subjected to isolation procedures within 96 h. The samples were washed thoroughly with running tap water and in ultrasonic bath to remove any adhering soil particles and airdried at ambient temperature for 48 h.

Sample Collection and Isolation of Endophytic
Two methods were employed for the isolation of the endophytic Actinobacteria using seven specific isolation media (Table 1).
The plant parts of D. cochinchinensis Lour. were excised and subjected to a five-step surface-sterilization procedure: a 4 min wash in 5% NaOCl, followed by 10 min wash in 2.5% Na 2 S 2 O 3 , a 5 min wash in 75% ethanol, a wash in sterile water, and a final rinse in 10% NaHCO 3 for 10 min. After drying thoroughly under sterile conditions, the surface sterilized tissues were disrupted aseptically in a commercial blender and distributed on isolation media [5,7].
Method 2. The surface sterilized plant parts (1-2 g) were sliced, grounded with mortar and pestle, and mixed with 0.5 g CaCO 3 . The samples were kept in a laminar flow cabinet for 14 d, incubated at 80 ∘ C for 30 min, and plated onto isolation media [7].
Each medium was supplemented with nalidixic acid (25 mg⋅L −1 ), nystatin (50 mg⋅L −1 ), and K 2 Cr 2 O 7 (50 mg⋅L −1 ) to inhibit the growth of Gram-negative bacteria and fungi; polyvinyl pyrrolidone (2%) and tannase (0.005%) were also added to improve the development of colonies on media. Colonies grown on these isolation media were selected and purified by repeated streaking on YIM 38 medium. The pure cultures were preserved as glycerol suspensions (20%, v/v) at −80 ∘ C and as lyophilized spore suspensions in skim milk (15%, w/v) at 4 ∘ C.

Identification and Diversity
Profiling. For phylogenetic characterization, genomics DNAs of all isolates were extracted using an enzyme hydrolysis method. About 50 mg of the freshly grown culture was taken in an autoclaved 1.5 mL Eppendorf tube. To the culture, 480 L TE buffer (1x) and 20 L lysozyme solution (2 mg⋅mL −1 ) were added. The bacterial suspension was thoroughly mixed and incubated for 2 h under shaking conditions (160 rpm, 37 ∘ C). The mixture was treated with 50 L SDS solution (20%, w/v) and 5 L Proteinase K solution (20 g⋅mL −1 ) and kept on a water bath (55 ∘ C, 1 h). DNA was then extracted twice with phenolchloroform-isoamyl alcohol (25 : 24 : 1 v/v/v), followed by precipitation with 80 L sodium acetate (3 mol⋅L −1 , pH 4.8-5.2) and 800 L absolute ethanol. The resulting DNA precipitate was centrifuged at 4 ∘ C (12,000 rpm, 10 min), washed with 70% ethanol, and then air-dried. The extracted DNA was resuspended in 30 L TE buffer and stored at −20 ∘ C. PCR amplification for 16S rRNA gene from the extracted DNA samples was done using the primer pair PA-PB (PA: 5 -CAGAGTTTGATCCTGGCT-3 ; PB: 5 -AGGAGGTGATCCAGCCGCA-3 ) as described previously [14]. Amplified PCR products were purified and sequenced by Sangon Biotech (Shanghai). Identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence similarities were achieved using the EzTaxon server (http://www.eztaxon.org/) [15] and BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The alignment of the sequences was done using CLUSTALW [16]. The phylogenetic tree was constructed using the aligned sequences by the neighbour-joining method [17] using Kimura 2-parameter distances [18] in the MEGA 6 software [19]. To determine the support of each clade, bootstrap analysis was performed with 1,000 replications [20].

Selection of Bioactive Actinobacteria Strains.
Each of the isolated Actinobacteria was screened for antimicrobial activity and anthracyclines production. The antibacterial activities were evaluated against Methicillin-resistant Staphylococcus epidermidis (MRSE) ATCC 35984, Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 25923, Methicillinsusceptible Staphylococcus aureus (MSSA) ATCC 29213, Klebsiella pneumoniae ATCC 13883, Aeromonas hydrophila ATCC 7966, and Escherichia coli ATCC 25922 using the agar well diffusion method [21]. Anthracycline productivity was screened using the pigment production test as described by Trease [22]. Based on the results of the two screenings, bioactive strains were selected for further assays.  [23,24]. These test pathogens were provided by CIRAD, UMR QUALISUD, France, and maintained on Potato Dextrose Agar (PDA). The cytotoxic activity of the selected strains was tested by sulforhodamine B (SRB) assay as described earlier [25][26][27]. The human breast adenocarcinoma (MCF-7) and human hepatocellular carcinoma (Hep G2) cells lines used for the test were procured from American Type Culture Collection (ATCC, Boulevard, Manassa, VA 20110, USA). Ellipticine was used as the positive control.

Screening for Biosynthetic
Genes. Three sets of PCR primers A3F/A7R, K1F/M6R, and KS F/KS R were used for amplification of nonribosomal peptide synthetase (NRPS), polyketide synthase-(PKS-) I, and PKS-II specific domains [6,28]. PCR amplifications were performed in a Biometra thermal cycler in a final volume of 25 L containing 0.2 mol⋅L −1 of each primer, 0.1 mol⋅L −1 of each of the four dNTPs (Takara, Japan), 2.5 L of extracted DNA, 0.5 unit of Taq DNA polymerase (with its recommended reaction buffer), and 10% of DMSO. Amplifications were performed according to the following profile: initial denaturation at 96 ∘ C for 5 min; 30 cycles of denaturation at 96 ∘ C for 1 min, primer annealing at either 57 ∘ C (for K1F/M6R, A3F/A7R) or 58 ∘ C (for KS F/KS R) for 1 min, and extension at 72 ∘ C for 1 min, followed by a final extension at 72 ∘ C for 5 min. The sizes of amplicons were 1,200-1,400 bp (K1F/M6R), 613 bp (KS F/KS R), and 700-800 bp (A3F/A7R).
During the present study, Method 2 was found to be more suitable for the isolation of endophytic Actinobacteria from tissues of D. cochinchinensis Lour. and accounted for nearly 65% of the total isolation. All the media used in the current study, except for sodium propionate-asparagine-salt agar, were suitable for isolation of endophytic Actinobacteria ( Figure 2).

Diversity
Profiling. Based on the 16S rRNA gene sequence analysis, the most abundant Actinobacteria genera were Streptomyces (86.84%), followed by Nocardiopsis (4.93%), , Nocardioides (0.33%), and Pseudonocardia (0.33%). The relative abundance of the endophytic Actinobacteria among the different sites is shown in Table 2. Among the different sampling sites, Yunnan and Ninh Binh yielded the highest diversity, each contributing eight genera of Actinobacteria. Yunnan samples yielded the genera Streptomyces,

Selection of Bioactive Actinobacteria Strains.
All 304 Actinobacteria isolates were tested for antimicrobial activity and anthracycline production. Table 3 represents the distribution of bioactive Actinobacteria. These bioactive strains were distributed in the genera Streptomyces, Nocardiopsis, Nocardioides, Pseudonocardia, and Tsukamurella. The genus Streptomyces possessed the highest proportion of isolates with antimicrobial activities. Anthracyclines are important group of antitumor antibiotics and are being used in cancer treatment [29,30]. Of the 304 strains, 49 strains tested positive for anthracycline production.

Evaluation of Antifungal and Cytotoxicity Effects of the Bioactive Strains.
Several strains among the selected bioactive Actinobacteria were positive for antifungal activities against the mycotoxins-producing F. graminearum, A. carbonarius, and A. westerdijkiae strains. Frequencies of the antifungal activities against the indicator fungal pathogens were as follows: F. graminearum: 58.8%; A. carbonarius: 41.2%; and A. westerdijkiae: 23.5%. Table 5 summarizes the antifungal profile of the selected 17 strains.
Of the 17 strains, three strains (HUST001, HUST004, and HUST005) exhibited cytotoxic effects against the two tested human cancer cell lines, MCF-7 and Hep G2 (Table 5). Strain HUST004 showed significant inhibition toward MCF-7 cells with IC 50 -value of 3 g⋅mL −1 , while strains HUST001 and HUST005 showed moderate activity with IC 50 -values of 19 and 25 g⋅mL −1 , respectively. Against Hep G2 cell lines, IC 50values for the strains HUST004 and HUST005 were 10 and 33 g⋅mL −1 , respectively. The remaining strains were inactive against the two cancer cell lines.

Screening of Biosynthetic
Genes. All 17 bioactive strains were investigated for the presence of PKS-I, PKS-II, and NRPS genes. Frequencies of positive PCR amplification of the three biosynthetic systems were 29.41%, 70.59%, and 23.53%, respectively ( Table 5). All these three genes were detected in two strains (HUST003, HUST004), which were identified as members of the genus Streptomyces. PKS-II gene was detected at highest frequencies in both Streptomyces and non-Streptomycetes genera, while PKS-I and NRPS genes were detected only in the genus Streptomyces.

Discussion
The plant source D. cochinchinensis is known for the production of dragon's blood [11]. Traditional practices of folk medicine involved extraction of dragon's blood from the plant. During its extraction, large scale exploitation of the plant is necessary owing to the low yield of plant's extract and slow growth of the plant, thereby resulting in destruction of large number of century old plant [13]. It is, therefore,    Many reports suggested that maximum endophytes were recovered from roots, followed by stems and leaves [9,[31][32][33][34]. Similar observation was found during our study whereby more number of isolates was obtained from roots than from stems or leaves (Table 6). This may be due to the fact that rhizospheric regions of the soil have higher concentration of nutrients. A report also suggested that microorganism enters various tissues of plant from rhizosphere and switched to endophytic lifestyles [40,41]. Isolation of more isolates using the second method may be attributed to the enrichment of the samples with calcium carbonate. Qin et al. [7] have reported that calcium carbonate altered the pH to alkaline conditions which favour the growth of Actinobacteria. Among various genera isolated, Streptomyces is predominantly present in the plant D. cochinchinensis. The finding is consistent with similar studies of endophytic bacteria [6,9,32,33,36]. In the present study, rare Actinobacteria of the genera Arthrobacter, Brevibacterium, Kocuria, Microbacterium, Nocardia, Nocardioides, Nocardiopsis, Pseudonocardia, Rhodococcus, and Tsukamurella were also isolated. Though Arthrobacter, Brevibacterium, Microbacterium, Nocardia, Nocardioides, Nocardiopsis, Pseudonocardia, Rhodococcus, and Tsukamurella have been reported as endophytic Actinobacteria of medicinal plant [6,7,[31][32][33][34], this study forms the first report for the isolation of Brachybacterium and Kocuria (Table 6).
In the study of Cui et al. [35], it was indicated that 71% of the fungal isolates obtained from D. cochinchinensis exhibited varied antitumor activities against five human cancer cell lines: HepG2, MCF7, SKVO3, Hl-60, and 293-T. Similarly, in the study of Khieu et al. [10], the compounds (Z)-tridec-7-3n3-1,2,13-tricarboxylic acid and Actinomycin-D produced by a Streptomyces sp. exhibited cytotoxic effect against two human cancer cell lines HepG2 and MCF-7. During the current study, three of the Streptomyces strains (HUST001, HUST004, and HUST005) produced potential cytotoxic activities. All the three studies on D. cochinchinensis indicated that the endophytic microbes associated with the plant are alternative sources for extraction of cytotoxic compounds. These studies further indicated that endophytic microbes can serve as a means for sustainable utilization of the plant resources by preserving the natural niche.
biosynthetic gene clusters. Despite this fact, positive reaction for the amplification of specific domains for the three biosynthetic gene clusters is an indirect indication for the presence of the biosynthetic gene. In the present study, 13 of the 17 bioactive strains were found to have at least one of the three biosynthetic gene clusters. Among them, strains HUST003 and HUST004 showed positive results for the presence of PKS-I, PKS-II, and NRPS genes and also exhibited antifungal activity against all test pathogens (Table 5). Strains HUST006, HUST008, and HUST017 were negative both for the presence of PKS-I, PKS-II, and NRPS genes and for antifungal activity. The results indicated that the antifungal metabolites of these bioactive strains might be products of these biosynthetic genes. Li et al. [4] and Qin et al. [7] had reported that number of isolates having antimicrobial property need not correlate with the percentage of isolates showing the presence of PKS and NRPS gene and vice versa. Strains HUST002, HUST009, HUST013, and HUST015 did not show any antifungal activity but they encoded at least one of these biosynthetic genes. Similarly strain HUST014 was absent for PKS or NRPS gene products but showed antifungal activity.

Conclusions
Relatively fewer studies have been done to explore the endophytic microbes associated with medicinal plant. This study showed that endophytic Actinobacteria associated with the medicinal plant D. cochinchinensis Lour. could be an alternate source for production of bioactive compounds that were previously obtained from the medicinal plant. It thereby provides a sustainable way of utilizing the medicinal plant without destroying the plant.

Conflicts of Interest
The authors declare that they have no conflicts of interest.