Endophytic Actinobacteria Associated with Dracaena cochinchinensis Lour.: Isolation, Diversity, and Their Cytotoxic Activities.
Actinobacteria, especially the genus Streptomyces, are major producers of bioactive metabolites  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 inner tissues of plants, especially those with ethnomedicinal value [4-10].
The plant Dracaena cochinchinensis Lour. has been used as a traditional folk medicine in the oriental countries including China . 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 . Apart from its medicinal use, it also finds applications as colouring materials and wood varnish . 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.
2. Materials and Methods
2.1. Sample Collection and Isolation of Endophytic Actinobacteria. Healthy plant samples (leaves, stems, and roots) of medicinal plant D. cochinchinensis Lour. were collected from four different provinces located in two countries: Pingxiang, Guangxi province, China (20[degrees]06702"N, 106[degrees]45701"E; elevation, 236 m); Xishuangbanna, Yunnan province, China (21[degrees]55,41,/N, 101[degrees]25/49"E; 984 m); Bach Ma National Park, Thua Thien Hue province, Vietnam (16[degrees]9,55"N, 107[degrees]55,19"E; 1450 m), and Cuc Phuong National Park, Ninh Binh province, Vietnam (20[degrees]19,8,,N, 105[degrees]37,20,,E; 338 m). 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 air-dried at ambient temperature for 48 h.
Two methods were employed for the isolation of the endophytic Actinobacteria using seven specific isolation media (Table 1).
Method 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.sub.2][S.sub.2][O.sub.3], a 5 min wash in 75% ethanol, a wash in sterile water, and a final rinse in 10% NaHC[O.sub.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 CaC[O.sub.3]. The samples were kept in a laminar flow cabinet for 14 d, incubated at 80[degrees]C for 30 min, and plated onto isolation media .
Each medium was supplemented with nalidixic acid (25mg x [L.sup.-1]), nystatin (50mg x [L.sup.-1]), and K2Cr2O7 (50mg x [L.sup.- 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[degrees] C and as lyophilized spore suspensions in skim milk (15%, w/v) at 4[degrees]C.
2.2. 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 [micro]L TE buffer (1x) and 20 [micro]L lysozyme solution (2mg x [L.sup.-1]) were added. The bacterial suspension was thoroughly mixed and incubated for 2h under shaking conditions (160 rpm, 37[degrees]C). The mixture was treated with 50 [micro]L SDS solution (20%, w/v) and 5 [micro]L Proteinase K solution (20 [micro]g x m[L.sup.-1]) and kept on a water bath (55[degrees]C, 1 h). DNA was then extracted twice with phenolchloroform- isoamyl alcohol (25: 24: 1 v/v/v), followed by precipitation with 80 [micro]L sodium acetate (3 mol x [L.sup.-1], pH 4.8-5.2) and 800 [micro]L absolute ethanol. The resulting DNA precipitate was centrifuged at 4[degrees]C (12,000 rpm, 10 min), washed with 70% ethanol, and then air-dried. The extracted DNA was resuspended in 30 [micro]L TE buffer and stored at -20[degrees] 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-37) as described previously . 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/)  and BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The alignment of the sequences was done using CLUSTALW . The phylogenetic tree was constructed using the aligned sequences by the neighbour-joining method  using Kimura 2-parameter distances  in the MEGA 6 software . To determine the support of each clade, bootstrap analysis was performed with 1,000 replications .
2.3. 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, Methicillin-susceptible Staphylococcus aureus (MSSA) ATCC 29213, Klebsiella pneumoniae ATCC 13883, Aeromonas hydrophila ATCC 7966, and Escherichia coli ATCC 25922 using the agar well diffusion method . Anthracycline productivity was screened using the pigment production test as described by Trease . Based on the results of the two screenings, bioactive strains were selected for further assays.
2.4. Antifungal and Cytotoxicity Tests. Antifungal activity of the selected bioactive strains was tested against Fusarium graminearum, Aspergillus carbonarius, and Aspergillus westerdijkiae (strains producing the mycotoxins deoxynivalenol and ochratoxin A) [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 [25-27]. 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.
2.5. Screening for Biosynthetic Genes. Three sets of PCR primers A3F/A7R, K1F/M6R, and KS[alpha]F/KS[alpha]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 [micro]L containing 0.2 [micro]mol x [L.sup.-1] of each primer, 0.1 [micro]mol x [L.sup.-1] of each of the four dNTPs (Takara, Japan), 2.5 [micro]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[degrees]C for 5 min; 30 cycles of denaturation at 96[degrees]C for 1min, primer annealing at either 57[degrees] C (for K1F/M6R, A3F/A7R) or 58[degrees]C (for KS[alpha]F/KS[alpha]R) for 1 min, and extension at 72[degrees]C for 1min, followed by a final extension at 72[degrees]C for 5 min. The sizes of amplicons were 1,200-1,400 bp (K1F/M6R), 613 bp (KSaF/KSaR), and 700-800 bp (A3F/A7R).
3.1. Isolation of Endophytic Actinobacteria. A total of 304 putative endophytic Actinobacteria were isolated from three different tissues of D. cochinchinensis Lour. The highest number of Actinobacteria was isolated from roots (117 strains, 38.49%), followed by stems (113 strains, 37.17%) and leaves (74 strains, 24.34%) (Figure 1). Among the sites, more Actinobacteria were isolated from Xishuangbanna (Yunnan province, China) and Cuc Phuong National Park (Ninh Binh province, Vietnam) (Figure 1).
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).
3.2. Diversity Profiling. Based on the 16S rRNA gene sequence analysis, the most abundant Actinobacteria genera were Streptomyces (86.84%), followed by Nocardiopsis (4.93%), Brevibacterium (1.64%), Microbacterium (1.64%), Tsukamurella (1.64%), Arthrobacter (0.66%), Brachybacterium (0.66%), Nocardia (0.66%), Rhodococcus (0.66%), Kocuria (0.33%), 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, Nocardiopsis, Brevibacterium, Microbacterium, Brachybacterium, Rhodococcus, Kocuria, and Tsukamurella, while Ninh Binh samples yielded Streptomyces, Tsukamurella, Nocardiopsis, Arthrobacter, Nocardia, Brevibacterium, Nocardioides, and Pseudonocardia. Thua Thien Hue samples contained Streptomyces, Nocardiopsis, and Microbacterium, while Streptomyces and Nocardiopsis were present in Guangxi samples.
3.3. 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.
Based on the results of the bioactivity screening, 17 strains (HUST001-HUST011, HUST013-HUST015, HUST017, HUST018, and HUST026) were selected for further antifungal and cytotoxicity studies (Table 4). Of the 17 strains, 14 belonged to the genera Streptomyces while the rest comprised Nocardioides, Nocardiopsis, and Pseudonocardia (Figure 3).
3.4. 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.sub.50]-value of 3 [micro]g x m[L.sup.-1], while strains HUST001 and HUST005 showed moderate activity with [IC.sub.50]-values of 19 and 25 [micro]g x m[L.sup.-1], respectively. Against Hep G2 cell lines, [IC.sub.50] values for the strains HUST004 and HUST005 were 10 and 33 [micro]g x m[L.sup.-1], respectively. The remaining strains were inactive against the two cancer cell lines.
3.5. 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 nonStreptomycetes genera, while PKS-I and NRPS genes were detected only in the genus Streptomyces.
The plant source D. cochinchinensis is known for the production of dragon's blood . 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 . It is, therefore, imperative to search for alternative source of the plant's metabolites to preserve the plant in its natural niche. One such means is to study the endophytic microbes associated with the plant. In an earlier study by Cui et al. , D. cochinchinensis collected from Beijing, China, had been used to study the endophytic fungal diversity. The study resulted in the isolation of 49 fungal strains distributed into 18 genera. In another study of endophytic microbe associated with D. cochinchinensis, Khieu et al.  had isolated a Streptomyces strain, producing two potent cytotoxic compounds, from plant samples collected from Cuc Phuong National Park, Ninh Binh province, Vietnam. But neither of these studies described the diversity profile of the Actinobacteria communities living in association with the plant. As endophytic Actinobacteria from medicinal plants have been a major research area in the search of new antibiotic-producing strains [4, 7, 8, 36-39], we have selected the same plant source for in-depth analysis of Actinobacteria community structure. The present study resulted in the isolation of 304 Actinobacteria strains.
Many reports suggested that maximum endophytes were recovered from roots, followed by stems and leaves [9, 31-34]. 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 thesecondmethodmaybeattributedto theenrichmentofthe samples with calcium carbonate. Qin et al.  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, 31-34], this study forms the first report for the isolation of Brachybacterium and Kocuria (Table 6).
Endophytic Actinobacteria are often associated with antimicrobial properties [6, 7, 31]. This is shown by the high proportion of antibacterial activities by endophytic Actinobacteria associated with D. cochinchinensis Lour.: 23.03% against ATCC 35984, 23.26% against ATCC 25923, 25% against ATCC 29213, 23.68% against ATCC 13883, 32.43% against ATCC 7966, and 17.43% against ATCC 25922. Based on the preliminary bioactivity profile, a set of 17 Actinobacteria were further studied for antifungal and cytotoxic properties. Of the 17 strains selected, 10 strains were significant against F. graminearum, seven against A. carbonarius, and four against A. westerdijkiae. Similar findings have been reported in related studies of Streptomyces strains [42-44]. Four strains (HUST003, HUST004, HUST005, and HUST026) showed remarkable antifungal activity against all test fungi (Table 5). In contrast to above strains, HUST002, HUST006, HUST008, HUST009, HUST013, HUST015, and HUST017 did not show any antifungal activity.
In the study of Cui et al. , 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. , the compounds (Z)-tridec-73n3-1,2,13- 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.
The cytotoxic abilities ([IC.sub.50]-values) of the three strains HUST001, HUST004, and HUST005 against the human cancer cell lines MCF-7 and/or Hep G2 range in between 3 and 33 [micro]g x m[L.sup.-1]. This finding is significant with reference to related studies [44-47]. Lu and Shen  isolated naphthomycin K from endophytic Streptomyces strain CS which exhibit cytotoxic activity against P388 and A-549 cell lines with [IC.sub.50]-values of 0.07 and 3.17 [micro]mol-[L.sup.-1]. Kim et al.  isolated salaceyins A and B from Streptomyces laceyi MS53 having [IC.sub.50]-values of 3.0 and 5.5 [micro]g x m[L.sup.-1] against human breast cancer cell line SKBR3.
The biosynthetic genes are involved in microbial natural product biosynthesis. The antitumor drug bleomycin from Streptomyces verticillus ATCC15003 involved a hybrid NRP-SPKS system . Genomic analysis of the specific strain will, however, be necessary for illustration of the presence of 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.  and Qin et al.  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 HUSTO02, 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.
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.
Nimaichand Salam and Thi-Nhan Khieu contributed equally to this work.
The authors are grateful to China Postdoctoral Science Foundation (Project no. 2016M602566), Visiting Scholar Grant of State Key Laboratory of Biocontrol, Sun Yat-Sen University (Project no. SKLBC14F02), Vietnam Ministry of Education and Training (Project no. B2014-01-79), and Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2014) for financial support for this study.
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Nimaichand Salam, (1) Thi-Nhan Khieu, (1,2) Min-Jiao Liu, (3) Thu-Trang Vu, (2) Son Chu-Ky, (2) Ngoc-Tung Quach, (4) Quyet-Tien Phi, (4) Manik Prabhu Narsing Rao, (1) Angelique Fontana, (5) Samira Sarter, (5) and Wen-Jun Li (1,3)
(1) State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China
(2) Department of Food Technology, School of Biotechnology and Food Technology, Hanoi University of Science and Technology, Hanoi, Vietnam
(3) Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, China
(4) Laboratory of Fermentation Technology, Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi, Vietnam
(5) CIRAD, UMR QUALISUD, 34398 Montpellier, France
Correspondence should be addressed to Son Chu-Ky; firstname.lastname@example.org and Wen- Jun Li; email@example.com
Received 30 June 2016; Accepted 20 March 2017; Published 6 April 2017
Academic Editor: Young-Mi Lee
Caption: Figure 3: Neighbour-joining phylogenetic dendrogram based on 16S rRNA gene sequences showing the relationship of the selected 18 endophytic Actinobacteria with their closest species.
Table 1: Composition of the seven media used for the isolation of endophytic Actinobacteria from Dracaena cochinchinensis Lour. Medium Name and composition (g [L.sup.-1] of water) Reference 1 Tap water-yeast extract agar (TWYE) [3,5] Yeast extract 0.25, [K.sub.2]HP[O.sub.4] 0.5, agar 15 2 Trehalose agar Trehalose 6, KN[O.sub.3] 0.5, C[aCl.sub.2]  0.3, [Na.sub.2]HP[O.sub.4] 0.3, MgS[O.sub.4] x 7[H.sub.2] O 0.2, agar 15 Sodium propionate agar  3 Sodium propionate 2, N[H.sub.4]N[O.sub.3] 0.1, KCl 0.1, MgS[O.sub.4] x 7[H.sub.2] O 0.05, FeS[O.sub.4] x 7[H.sub.2] O 0.05, agar 15 Starch agar  4 Starch 2, KN[O.sub.3]1, NaCl 0.4, [K.sub.2]HP[O.sub.4] 0.5, MgS[O.sub.4] x 7[H.sub.2] O 0.5, FeS[O.sub.4] x 7[H.sub.2] O 0.01, agar 15 Citrate agar This study 5 Citric acid 0.12, ferric ammonium citrate 0.12, NaN[O.sub.3] 1.5, [K.sub.2] HP[O.sub.4] x 3[H.sub.2] O 0.4, MgS[O.sub.4] x 7[H.sub.2] O 0.1, Ca[Cl.sub.2] x [H.sub.2]O 0.05, EDTA 0.02, [Na.sub.2]C[O.sub.3] 0.2, agar 15 Sodium propionate-asparagine-salt agar 6 Sodium propionate 4, asparagine 1, casein 2,  [K.sub.2]HP[O.sub.4] 1, MgS[O.sub.4] x 7[H.sub.2] O 0.1, FeS[O.sub.4] x 7[H.sub.2] O 0.01, NaCl 30, agar 15 Dulcitol-proline agar This study 7 Dulcitol 2, proline 0.5, [K.sub.2]HP[O.sub.4] 0.3, NaCl 0.3, MgS[O.sub.4] x 7[H.sub.2] O 1, Ca[Cl.sub.2] x 2[H.sub.2] O 1, agar 15 Table 2: Distribution of endophytic Actinobacteria isolated from the different tissues of D. cochinchinensis Lour. among the different sampling sites. Genera Yunnan Guangxi Thua Thien Hue Ninh Binh China China Vietnam Vietnam Arthrobacter 0 0 0 2 Brachybacterium 2 0 0 0 Brevibacterium 4 0 0 1 Kocuria 1 0 0 0 Microbacterium 4 0 1 0 Nocardia 0 0 0 2 Nocardioides 0 0 0 1 Nocardiopsis 8 1 2 4 Pseudonocardia 0 0 0 1 Rhodococcus 2 0 0 0 Streptomyces 104 46 30 82 Tsukamurella 1 0 0 5 Total 126 47 33 98 Genera Total Arthrobacter 2 Brachybacterium 2 Brevibacterium 5 Kocuria 1 Microbacterium 5 Nocardia 2 Nocardioides 1 Nocardiopsis 15 Pseudonocardia 1 Rhodococcus 2 Streptomyces 262 Tsukamurella 6 Total 304 Table 3: Bioactivity profiles of the endophytic Actinobacteria i solated from D. cochinchinensis Lour. Genera Antimicrobial activity ATCC ATCC ATCC ATCC ATCC ATCC 35984 25923 29213 13883 7966 25922 Arthrobacter 0 0 0 0 0 0 Brachybacterium 0 0 0 0 0 0 Brevibacterium 0 0 0 0 0 0 Kocuria 0 0 0 0 0 0 Microbacterium 0 0 0 0 0 0 Nocardia 0 0 0 0 0 0 Nocardioides 0 0 1 1 0 0 Nocardiopsis 0 3 4 1 0 0 Pseudonocardia 0 0 1 0 1 0 Rhodococcus 0 0 0 0 0 0 Streptomyces 70 68 70 70 96 53 Tsukamurella 0 0 0 0 1 0 Total 70 71 76 72 98 53 Proportion (%) 23.03 23.26 25.00 23.68 32.43 17.43 Genera Anthracycline production Arthrobacter 0 Brachybacterium 0 Brevibacterium 0 Kocuria 0 Microbacterium 0 Nocardia 0 Nocardioides 1 Nocardiopsis 1 Pseudonocardia 1 Rhodococcus 0 Streptomyces 46 Tsukamurella 0 Total 49 Proportion (%) 16.11 Note. Number indicates number of isolates positive for the particular bioactivity. ATCC 35984, Methicillin-resistant Staphylococcus epidermidis (MRSE); ATCC 25923, Methicillin-resistant Staphylococcus aureus (MRSA); ATCC 29213, Methicillin-susceptible Staphylococcus aureus (MSSA); ATCC 13883, Klebsiella pneumoniae; ATCC 7966, Aeromonas hydrophila; ATCC 25922, Escherichia coli. Table 4: Isolation and characterization profile of the 17 selected endophytic Actinobacteria. Strain Sampling Isolation Isolation Source Accession site* medium method number HUST001 NB 3 2 Stem KT033860 HUST002 GX 2 1 Stem KP317660 HUST003 TTH 5 1 Stem KT033861 HUST004 YN 3 2 Root KT033862 HUST005 NB 4 2 Stem KT033863 HUST006 NB 3 2 Stem KT033864 HUST007 YN 5 1 Root KT033865 HUST008 TTH 6 2 Stem KT033866 HUST009 YN 3 2 Stem KT033867 HUST010 YN 2 1 Root KT033868 HUST011 GX 3 1 Root KT033869 HUST013 NB 4 1 Root KT033870 HUST014 TTH 5 1 Root KT033871 HUST015 TTH 7 2 Stem KT033872 HUST017 YN 2 2 Leaf KT033873 HUST018 NB 1 2 Root KT033874 HUST026 NB 1 2 Root KT033859 Strain Closest homologs HUST001 Streptomyces puniceus NBRC 12811T HUST002 Streptomyces violarus NBRC 13104T HUST003 Streptomyces cavourensis NBRC 13026T HUST004 Streptomyces cavourensis NBRC 13026T HUST005 Streptomyces parvulus NBRC 13193T HUST006 Streptomyces rubiginosohelvolus NBRC 12912T HUST007 Streptomyces puniceus NBRC 12811T HUST008 Streptomyces puniceus NBRC 12811T HUST009 Streptomyces puniceus NBRC 12811T HUST010 Streptomyces pluricolorescens NBRC 12808T HUST011 Streptomyces parvulus NBRC 12811T HUST013 Pseudonocardia carboxidivorans Y8T HUST014 Streptomyces augustmycinicus NBRC 3934T HUST015 Streptomyces violarus NBRC 13104T HUST017 Nocardiopsis dassonvillei subsp. albirubida DSM 40465t HUST018 Streptomyces graminisoli JR- 19T HUST026 Nocardioides ganghwensis JC2055t Strain Pairwise similarity HUST001 100.0 HUST002 99.45 HUST003 99.70 HUST004 100.0 HUST005 99.73 HUST006 99.72 HUST007 100.0 HUST008 99.80 HUST009 98.66 HUST010 100.0 HUST011 100.0 HUST013 100.0 HUST014 99.85 HUST015 99.57 HUST017 100.0 HUST018 99.45 HUST026 98.26 * YN, Xishuangbanna, Yunnan province, China; GX, Pingxiang, Guangxi province, China; TTH, Bach MaNational Park, Thua Thien Hue province, Vietnam; NB, Cuc Phuong National Park, Ninh Binh province, Vietnam. Table 5: Antifungal, cytotoxic, and biosynthetic gene profiles of the 17 selected endophytic Actinobacteria isolated from D. cochinchinensis Lour. Test pathogens Strain Fusarium Aspergillus Aspergillus graminearum carbonarius westerdijkiae HUST001 + - - HUST002 - - - HUST003 + + + HUST004 + + + HUST005 + + + HUST006 - - - HUST007 + - - HUST008 - - - HUST009 - - - HUST010 + + - HUST011 + + - HUST013 - - - HUST014 + - - HUST015 - - - HUST017 - - - HUST018 + + - HUST026 + + + Cytotoxicity on MCF-7 (given in % inhibition) Strain Concentration ([micro]g x [ml.sup.-1]) 10000 2000 400 80 16 [IC.sub.50] HUST001 101.74 94.48 70.47 63.70 48.84 19 HUST002 112.12 89.42 62.60 31.86 15.99 194 HUST003 106.71 97.45 67.50 44.38 19.33 120 HUST004 105.40 88.68 73.52 62.87 56.94 3 HUST005 107.96 106.95 103.59 58.13 44.02 25 HUST006 78.54 17.16 5.59 -1.08 -10.50 5710 HUST007 97.06 82.03 33.40 25.28 18.73 832 HUST008 99.71 81.92 42.76 30.32 11.34 399 HUST009 94.51 84.24 26.54 16.43 8.34 870 HUST010 98.86 98.72 68.98 29.28 11.93 166 HUST011 98.10 54.71 47.71 39.48 24.42 695 HUST013 53.93 0.62 -3.91 -5.34 -6.81 9517 HUST014 91.67 80.05 52.38 43.63 11.27 249 HUST015 99.05 83.68 70.60 37.20 15.59 129 HUST017 22.66 -0.40 -1.00 -2.63 -8.75 >10000 HUST018 42.02 6.29 4.41 -1.26 0.57 >10000 HUST026 92.08 86.55 37.72 28.64 22.61 623 Cytotoxicity on Hep G2 (given in % inhibition) Strain 10000 2000 400 80 16 [IC.sub.50] HUST001 103.92 103.29 102.75 56.25 12.42 68 HUST002 105.66 94.93 45.31 17.74 4.48 547 HUST003 109.97 109.29 90.54 54.56 -4.41 56 HUST004 109.18 107.70 103.23 87.50 62.08 10 HUST005 109.38 95.95 97.89 56.33 37.92 33 HUST006 88.18 13.60 9.25 -2.98 -5.14 5745 HUST007 125.34 107.26 35.64 -3.78 -5.46 587 HUST008 105.41 103.72 27.53 8.78 -2.05 633 HUST009 121.62 104.90 18.83 -6.83 -17.04 688 HUST010 98.50 97.87 58.09 34.39 13.29 271 HUST011 85.25 47.37 27.53 13.06 -14.29 1721 HUST013 9.34 -3.28 -14.36 -19.50 -13.38 >10000 HUST014 109.90 106.17 86.86 46.85 -6.02 77 HUST015 101.49 97.87 76.25 32.02 -4.37 172 HUST017 -1.27 -1.70 -2.76 -1.54 -12.18 >10000 HUST018 32.18 -0.88 -2.25 -12.44 -15.58 >10000 HUST026 104.63 86.23 38.05 21.95 10.72 691 Biosynthetic genes Strain PKS-I PKS-II NRPS HUST001 - + - HUST002 - + - HUST003 + + + HUST004 + + + HUST005 - + + HUST006 - - - HUST007 + - - HUST008 - - - HUST009 + + - HUST010 - + - HUST011 + + - HUST013 - + - HUST014 - - - HUST015 - + - HUST017 - - - HUST018 - + + HUST026 - + - Table 6: Comparative endophytic Actinobacteria diversity profile from different plant sources. Number of isolates from Plant sources different tissues Leaves Roots Stems Others Artemisia annua / / / / (Yunnan, China) Maytenus austroyunnanensis 102 126 84 / (Yunnan, China) 36 plant species (Chiang Mai, 97 212 21 / Thailand) Azadirachta indica A. Juss. (Varanasi, 12 30 13 / India) 7 plant species 6 22 9 2 (Mizoram, India) 26 species 78 326 156 / (Sichuan, China) Dracaena cochinchinensis Lour. (China and 74 117 113 / Vietnam) Plant sources Diversity profile* Reference Streptomyces (123); Promicromonospora (26); Pseudonocardia (15); Nocardia (11); Artemisia annua Nonomuraea (10); Rhodococcus (8);  (Yunnan, China) Kribbella (7); Micromonospora (7); Actinomadura (6); Amycolatposis (3); Streptosporangium (3); Dactylosporangium (2); Blastococcus (1); Glycomyces (1); Gordonia (1); Kocuria (1); Microbispora (1); Micrococcus (1); Phytomonospora (1) Streptomyces (208); Pseudonocardia (22); Nocardiopsis (21); Micromonospora (17); Maytenus Promicromonospora (6); austroyunnanensis Streptosporangium (6); Actinomadura  (Yunnan, China) (4); Amycolatopsis (4); Nonomuraea (4); Mycobacterium (3); Glycomyces (2); Gordonia (2); Microbacterium (2); Plantactinospora (2); Saccharopolyspora (2); Tsukamurella (2); Cellulosimicrobium (1); Janibacter (1); Jiangella (1); Nocardia (1); Polymorphospora (1) 36 plant species Streptomyces (277); Microbispora (Chiang Mai, (14); Nocardia (8); Micromonospora  Thailand) (4); uncharacterized (27) Azadirachta indica Streptomyces (27); A. Juss. (Varanasi, Streptosporangium (8); Microbispora  India) (6); Streptoverticillium (3); Saccharomonospora (3); Nocardia (2) 7 plant species Streptomyces (23); Microbacterium  (Mizoram, India) (9); Leifsonia (1); Brevibacterium (1); Uncharacterized (3) 26 species Streptomyces, Micromonospora,  (Sichuan, China) Nonomuraea, Oerskovia, Promicromonospora, Rhodococcus Dracaena Streptomyces (264); Nocardiopsis cochinchinensis (15); Brevibacterium (5); This study Lour. (China and Microbacterium (5); Tsukamurella Vietnam) (5); Arthrobacter (2); Brachybacterium (2); Nocardia (2); Rhodococcus (2); Kocuria (1); Nocardioides (1); Pseudonocardia (1) * Number within parentheses indicates the number of strains from each genera; / indicates no data. Figure 1: Distribution of endophytic Actinobacteria isolated from the different tissues of Dracaena cochinchinensis Lour. among the different sampling sites. Roots Stems Leaves Yunnan 48 44 34 Guangxi 14 18 15 Thua Thien Hue 3 18 12 Ninh Binh 52 33 13 Total 117 113 74 Note: Table made from bar graph. Figure 2: Effect of media on the isolation of endophytic Actinobacteria. Medium 1 11% Medium 2 16% Medium 3 19% Medium 4 18% Medium 5 18% Medium 6 3 Medium 7 15% Note: Table made from pie chart.
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|Title Annotation:||Research Article|
|Author:||Salam, Nimaichand; Khieu, Thi-Nhan; Liu, Min-Jiao; Vu, Thu-Trang; Chu-Ky, Son; Quach, Ngoc-Tung; Phi|
|Publication:||BioMed Research International|
|Date:||Jan 1, 2017|
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