Diversity, bioactivities, and metabolic potentials of endophytic actinomycetes isolated from traditional medicinal plants in Sichuan, China

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1 Chinese Journal of Natural Medicines 2015, 13(12): Chinese Journal of Natural Medicines Diversity, bioactivities, and metabolic potentials of endophytic actinomycetes isolated from traditional medicinal plants in Sichuan, China QIU Peng 1, 2, FENG Zhi-Xiang 1, 3, TIAN Jie-Wei 1, 2, LEI Zu-Chao 1, 2, WANG Lei 1, 2, ZENG Zhi-Gang 3, CHU Yi-Wen 3 1, 2*, TIAN Yong-Qiang 1 Key laboratory of Leather Chemistry and Engineering, Ministry of Education and College of Light Industry, Textile & Food Engineering, Sichuan University, Chengdu, , China; 2 Department of Pharmaceutical and Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu , China; 3 Sichuan Industrial Institute of Antibiotics, Chengdu University, Chengdu , China Available online 20 Dec., 2015 [ABSTRACT] The present study was designed to determine the taxonomic diversity and metabolic activity of the actinomycetes community, including 13 traditional medicinal plants collected in Sichuan province, China, using multiple approaches such as morphological and molecular identification methods, bioactivity assays, and PCR screening for genes involved in antibiotics biosynthesis. 119 endophytic actinomycetes were recovered; 80 representative strains were chosen for 16S rrna gene partial sequence analyses, with 66 of them being affiliated to genus Streptomyces and the remaining 14 strains being rare actinomycetes. Antimicrobial tests showed that 12 (15%) of the 80 endophytic actinomycetes displayed inhibitory effects against at least one indicator pathogens, which were all assigned to the genus Streptomyces. In addition, 87.5% and 58.8% of the isolates showed anticancer and anti-diabetic activities, respectively. Meanwhile, the anticancer activities of the isolates negatively correlated with their anti-diabetic activities. Based on the results of PCR screening, five genes, PKS-I, PKS-II, NRPS, ANSA, and oxyb, were detected in 55.0%, 58.8%, 90.0%, 18.8% and 8.8% of the 80 actinomycetes, respectively. In conclusion, the PCR screening method employed in the present study was conducive for screening and selection of potential actinomycetes and predicting potential secondary metabolites, which could overcome the limitations of traditional activity screening models. [KEY WORDS] Endophytic actinomycetes; Medicinal plants; Diversity; Bioactivity; PCR screening [CLC Number] R931.2 [Document code] A [Article ID] (2015) Introduction Actinomycetes are known as the world s greatest reservoir of natural antibiotics [1], but the frequency of discovering structurally new bioactive compounds is [Received on] 13-Jan [Research funding] This work was supported by Sichuan Science and Technology Project (No. 2014JY0199) and the Fundamental Research Funds for Central University (SCU2005D008). [ * Corresponding author] Tel/Fax: , These authors contributed equally to this work. These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved apparently decreasing in recent years [2-3]. Endophytic actinomycetes have been proven to be a new untapped source of novel actinomycetes [4]. Endophytes have been confirmed to have diverse intimate associations with hosts, such as nitrogen fixation, biosynthetic gene exchange, and production of phytohormones [5-8]. Several new strategies have been successfully applied to find new species or novel chemical candidates with potential bioactivities, including the utilization of edna [9] and combinational biosynthesis [10]. It is also meaningful to search and exploit endophytic microorganisms. The unique interior niches of endophytic actinomycetes have attracted extensive attention in developing novel natural bioactive compounds or unusual biochemical mechanism to relieve problems in rare-resource substitution and cope with drug-resistance situations [11-12]. 942

2 The selection rationale of host plants for endophytes isolation is important. The plants growing in unique environments or special endemic locations could produce novel endophytic microorganisms [12]. Ethnobotany history has shown their potential medicinal uses. For example, the plants such as Kennedia nigriscans, have been used as traditional medicines, from which a broad-spectrum antibiotic (munumbicins) has been discovered, and kakadumycins are isolated from some endophytes of Grevillea pteridifolia [13-15]. Natural products screening has been relied on the use of biologically activity models. More recently, pre-evaluation on biosynthetic potential of bioactive metabolites could reduce the workload by focusingon the most metabolically talented groups [16-19]. In the present study, we investigated the endophytic actinomycetes from 13 traditional medicinal or ethnomedical plants collected in Qingcheng and Emei Mountains of Sichuan province, China, where more than species of traditional medicinal plants have been reported. By screening for antimicrobial, anticancer, and anti-diabetic activities and biosynthesis potentials of these endophytic actinomycetes, we hoped to discover some prominent candidates for further research and application in pharmaceutical and agricultural industries. Materials and Methods Cells, chemicals, and reagents Stem and root materials of 13 healthy native traditional medicinal plants were collected from Qingcheng Mountain ( E, N) and Emei Mountain ( E, N) in Sichuan, China (Table 1), between April 2008 and April This study didn t need specific permissions, because the sample collection did not involve any endangered or protected species. The cut edges of the plants were sealed with Parafilm TM (Polysciences Inc, USA), and rhizosphere soils on the roots were removed with brush. All the samples were labeled, stored in clean envelopes, and then brought to the laboratory for isolation of endophytic actinomycetes within 48 h after collection. The human cancer cells HepG2, plasmid pppre-luc and phrl-tk were donated by Institute of Materia Medica of Diao Pharmaceutical Co, (Chengdu, China). All the indicator strains Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Aspergillus fumigatus, Candida albicans, Aspergillus niger, and Gibberalla fujikuroi were provided by Microbial Resources Center (MRC) of Sichuan Industrial Institute of Antibiotics (SIIA, Chengdu, China). Vitamins of lactoflavine, thiamine, bioepiderm, nicotinamide, calcium pantothenate, cyclohexanhexol, pyridoxine for medium preparation were purchase from Sangon Biotech., (Shanghai, China). Glucose, oat meal, dextrin, peptone, fish meal, yeast extract, agar powder, n-butanol, alcohol, NaClO and DMSO were purchased from Changzhen Co. (Chengdu Chian). Antibiotics nalidixic acid, cyclohexmide, streptomycin and penicillin used in this study were purchased from Sangon Biotech., (Shanghai, China). Taq DNA polymerase and dntp for PCR amplication were purchased from Takara Co., Ltd., (Dalian, China). Primers used in this study were synthesized by Sangon Biotech., (Shanghai, China). Isolation of endophytic actinomycetes After a 24-h air drying, the samples were pretreated using the procedure as follows: wiping the cut edges of stems and roots with 75% ethanol; washing thoroughly to remove soil and trashes; and dislodging any soil particles and epiphytes from the samples using an ultrasonic cleaner for 5 min. After an air drying in sterile condition, the samples were surface-sterilized using a four-step procedure as follows: a 60-s wash in 99% (V/V) ethanol, a 6-min wash in 3.125% (available chlorine) NaClO, a 30-s wash in 99% (V/V) ethanol, and a final rinse in sterile distilled water four times. To validate the effectiveness of the surface-sterilization procedure, 0.5 ml of the last rinsing water was spread on a blank control plate. The surfacesterilized materials were aseptically sectioned into small pieces (0.5 cm 0.8 cm), distributed on tap water-yeast extract agar (TWYE) [20] and T-improved agar, and then incubated at 28 ± 2 o C for days to record microbial growth. After incubation, clones were picked for identification. Nalidixic acid (25 μg ml 1 ) and cyclohexmide (50 μg ml 1 ) were added in the medium to suppress the growth of non-actinomycetes bacteria and fungi. T-improved agar used in the present study was an improved formula based on TWYE agar by adding several growth factors, including 0.5 mg lactoflavine, 0.5 mg thiamine, 0.25 mg bioepiderm, 0.5 mg nicotinamide, 0.5 mg calcium pantothenate, 0.5 mg cyclohexanhexol, 0.5 mg pridoxine, and 0.5 mg para-aminobenzoic acid per ml medium. Taxonomic affiliation and phylogenetic analysis based on partial 16S rrna gene sequence The isolated strains were purified and tentatively identified as different groups based on their cultural and morphological characteristics on Gause s synthetic agar, ISP3, and ISP2 media [21]. The presence of aerial mycelia, spore mass color, diffusible pigment color, and sporophore and spore chain morphology were used to characterize different groups, according to the Bergey s manual of systematic bacteriology [22]. The 16S rrna genes (610 bp, partial sequence) of 80 representative isolates from morphologically different groups were analyzed by direct PCR method with 2 pairs of primers [23]. The PCR primers were SRR181 Forward (5 -GTT TGA TCC TGG CTC AGG AC-3 ), SRR182 Reverse (5 -GGT GTT CCT CMH GAT ATC TG-3 ), SRR178 Forward (5 -GAA CGC TGG CGG CGT GCT-3 ), and SRR179 Reverse (5 -GCG CAT TYC ACC GCT ACA CC-3 ). The mixture of the PCR reactions (25 μl) contained the following: 1 PCR buffer (Takara), 2.5 mmol L 1 MgCl 2, 0.3 mmol L 1 dntps; 1 μmol L 1 of each primer, 1.25 U Taq DNA polymerase (Takara Co., Ltd., Dalian, China,), and 1 μl colony suspension (DNA template). The PCR cycle included the following: (1) an initial denaturation step for 4 min at 97 o C; (2) 35 cycles of 30 s at 97 o C, 30-s annealing between 58 and 65 o C, 943

3 and 45-s extension at 72 o C; and (3) a final extension step at 72 o C for 10 min. Aliquots of the 16S rrna gene amplicons (5 μl) were visualized by 1% agarose gel electrophoresis stained with ethidium bromide. Sequencing was performed by Invitrogen TM Biotechnology, Ltd. (Shanghai, China). Phylogenetic analysis of strains from the 13 medicinal plants was investigated. Their sequences were deposited in GenBank, their accession numbers were listed in Table 1. The partial sequence of 16S rrna gene of Escherichia coli was used as outgroup in neighbor-joining phylogenetic analysis. The sequences were first submitted to National Center for Biotechnology Information (NCBI) to obtain the closest phylogenetic relatives in GenBank database (http://www.ncbi. nlm.nih.gov/) using BLASTn version [24]. All the sequences were then aligned in Clustal W [25] and edited into a multiple alignment file in BioEdit version 1.0 [26]. The neighbor-joining phylogenetic tree was finally constructed with MEGA version 4.0 bootstrap analysis [27]. Fermentation and extraction The strains were cultured in a 50-mL shake-flask system of Table 1 The 80 isolates and their respective host plants Strain No. Host Accession No. Strain No. Host Accession No. A210 Perrottetia racemosa (Oliv.) Loes. JQ A255 Forsythia suspensa (Thunb.) Vahl HQ A211 Perrottetia racemosa (Oliv.) Loes. JQ A256 Forsythia suspensa (Thunb.) Vahl HQ A212 Perrottetia racemosa (Oliv.) Loes. JQ A257 Forsythia suspensa (Thunb.) Vahl HQ A213 Perrottetia racemosa (Oliv.) Loes. JQ A258 Forsythia suspensa (Thunb.) Vahl HQ A215 Celastrus rugosus Rehd. et Wils. JQ A259 Forsythia suspensa (Thunb.) Vahl HQ A217 Dioscorea bulbifera Linn. JF A260 Forsythia suspensa (Thunb.) Vahl HQ A219 Dioscorea bulbifera Linn. JF A261 Forsythia suspensa (Thunb.) Vahl HQ A220 Dioscorea bulbifera Linn. JF A262 Forsythia suspensa (Thunb.) Vahl HQ A221 Dioscorea bulbifera Linn. JF A263 Forsythia suspensa (Thunb.) Vahl HQ A222 Dioscorea bulbifera Linn. JF A266 Rumex dentatus Linn. JQ A223 Dioscorea bulbifera Linn. JF A267 Rumex dentatus Linn. JQ A224 Dioscorea bulbifera Linn. JF A268 Rumex dentatus Linn. JQ A225 Dioscorea bulbifera Linn. JF A269 Rumex dentatus Linn. JQ A226 Dioscorea bulbifera Linn. JF A270 Cinnamomum camphora (Linn.) Presl JQ A227 Dioscorea bulbifera Linn. JF A274 Solanum torvum Swartz. HQ A228 Dioscorea bulbifera Linn. JF A275 Solanum torvum Swartz. HQ A229 Dioscorea bulbifera Linn. JF A276 Solanum torvum Swartz. HQ A230 Dioscorea bulbifera Linn. JF A277 Solanum torvum Swartz. HQ A231 Chenopodium album Linn. JF A279 Dioscorea bulbifera Linn. JF A233 Chenopodium album Linn. JF A280 Dioscorea bulbifera Linn. JF A234 Chenopodium album Linn. JF A281 Dioscorea bulbifera Linn. JF A235 Chenopodium album Linn. JF A288 Impatiens nolitangere Linn. JQ A236 Chenopodium album Linn. JF A289 Impatiens nolitangere Linn. JQ A237 Chenopodium album Linn. JF A290 Impatiens nolitangere Linn. JQ A238 Chenopodium album Linn. JF A291 Impatiens nolitangere Linn. JQ A239 Chenopodium album Linn. JF A301 Geranium carolinianum Linn. JQ A241 Podocarpus macrophyllus (Thunb.) Sweet HM A315 Aconitum carmichaeli Debx. JQ A242 Podocarpus macrophyllus (Thunb.) Sweet HM A316 Aconitum carmichaeli Debx. JQ A243 Podocarpus macrophyllus (Thunb.) Sweet HM A317 Aconitum carmichaeli Debx. JQ A244 Podocarpus macrophyllus (Thunb.) Sweet HM A318 Aconitum carmichaeli Debx. JQ A245 Podocarpus macrophyllus (Thunb.) Sweet HM A319 Aconitum carmichaeli Debx. JQ A246 Populus adenopoda Maxim. HM A320 Aconitum carmichaeli Debx. JQ A247 Populus adenopoda Maxim. HM A321 Aconitum carmichaeli Debx. JQ A248 Populus adenopoda Maxim. HM A322 Aconitum carmichaeli Debx. JQ A249 Populus adenopoda Maxim. HM A323 Aconitum carmichaeli Debx. JQ A250 Populus adenopoda Maxim. HM A324 Aconitum carmichaeli Debx. JQ A251 Populus adenopoda Maxim. HM A325 Aconitum carmichaeli Debx. JQ A252 Populus adenopoda Maxim. HM A326 Chenopodium album Linn. JF A253 Populus adenopoda Maxim. HM A327 Chenopodium album Linn. JF A254 Forsythia suspensa (Thunb.) Vahl HQ A328 Chenopodium album Linn. JF

4 fermentation medium containing 10 g glucose, 20 g oat meal, 25 g dextrin, 10 g peptone, 5 g fish meal, 2 g yeast extract, and 3 g CaCO 3 in1 000 ml distilled water (ph ) at 28 ± 2 o C for 7 days, with continuous shaking at 200 r min 1. After fermentation, each individual culture broth was extracted with 25 ml of n-butanol for 30 min. The organic solvent extract was separated after centrifugation (2 000 g) for 10 min, concentrated with rotary evaporators, and cryodesiccated into arid extract, which was quantified for further bioactivity screening analysis. Screening of antimicrobial activity Bacterial and fungal indicator microorganisms were cultured on LB agar and fresh potato dextrose agar (PDA) media, respectively. Ten indicators were used, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Aspergillus fumigatus, Candida albicans, Aspergillus niger, and Gibberalla fujikuroi. All the indicator strains were provided by Microbial Resources Center (MRC) of Sichuan Industrial Institute of Antibiotics (SIIA, Chengdu, China). The assay was repeated twice. The inhibitory effects of the samples were determined using a modified double layer technique with 5-mm paper discs [28]. The arid extract samples were individually dissolved in DMSO at a concentration of 100 mg ml 1, and then diluted to 100 μg ml 1 as test-solutions with aseptic distilled water. Microbe suspension (for bacteria) or spores suspension (for fungi) was mingled with LB or PDA medium, and the final concentrations were cells/ml. The testsolution (10 μl) was added onto each filter paper disc placed on the surface of assay plates. After incubation at 37 o C (for bacteria) or at 28 o C (for fungi) for h, the diameter Φ of inhibition zones around each disc were measured. The antimicrobial activity was qualitatively evaluated as very strong if Φ was greater than 15 mm. The evaluation scale is shown in Table 2. Table 2 Partial results of bioactivity and phyla identification of isolates from 13 traditional medicinal plants Representa tive strain no. Genus Host Cytotoxic activity Anti-diabete s activity Klebsiella Pseudomona pneumoniae s aeruginosa Staphylococ cus aureus Staphylococcus Bacillus epidermidis subtilis A211 Streptomyces sp. Perrottetia racemosa (Oliv.) Loes A215 Streptomyces sp. Celastrus rugosus Rehd. et Wils A217 Streptomyces sp. Dioscorea bulbifera Linn A221 Streptomyces sp. Dioscorea bulbifera Linn A224 Streptomyces sp. Dioscorea bulbifera Linn A228 Streptomyces sp. Dioscorea bulbifera Linn A229 Streptomyces sp. Dioscorea bulbifera Linn A235 Actinocorallia sp. Chenopodium album Linn A236 Streptomyces sp. Chenopodium album Linn A238 Streptomyces sp. Chenopodium album Linn A243 Streptomyces sp. Podocarpus macrophyllus (Thunb.) Sweet + + A245 Podocarpus macrophyllus Actinomadura sp. (Thunb.) Sweet A246 Nocardia sp. Populus adenopoda Maxim A248 Microtetraspora sp. Populus adenopoda Maxim A260 Streptomyces sp. Forsythia suspensa (Thunb.) Vahl A263 Streptomyces sp. Forsythia suspensa (Thunb.) Vahl A267 Streptomyces sp. Rumex dentatus Linn A270 Streptomyces sp. Cinnamomum camphora (Linn.) Presl A277 Streptomyces sp. Solanum torvum Swartz A280 Streptomyces sp. Dioscorea bulbifera Linn A281 Streptomyces sp. Dioscorea bulbifera Linn A290 Micromonospora sp. Impatiens nolitangere Linn ( ) no inhibition, (+) inhibition zone or activity intensity; ( ) represent diameter of growth inhibition zone (Φ) > 15 mm, growth rate (GR) < 50, excitation rate (ER) > 100; ( ) Φ: mm, GR: 50 70, ER: ; (+ + +) Φ: mm, GR: 70 80, ER: 30 50; (+ +) Φ: mm, GR > 80, ER < 30 Screening of anticancer and anti-diabetic activities The in vitro anticancer activity of the culture extract was tested with the SRB assay [29] in human cancer cell line HepG2. After cells were seeded in 96-well plates at a density 945

5 of /well, added tested sample to cell suspension. All the samples were repeatedly tested twice at 6 grade from 100 ng ml 1 to 10 μg ml 1. The no-growth control (day 0) containing only cell suspension was set aside and the remaining assay plates were incubated at 37 o C for 72 h. After cell fixation and staining, the cell viability of HepG2 was tested by measuring optical density (OD) at 490 nm using a 96-well microtiter plate reader (VICTOR TM, Perkin-Elmer, USA). The cell growth rate was calculated by the following equation: if OD sample OD day0, growth rate = (OD sample OD day0 )/(OD neg control OD day0) 100%, and if OD sample < OD day0, cells killed = (OD day0 OD sample )/OD day0 100% [30]. The anticancer activity was considered as strong when growth rate was below 30%. Anti-diabetic activity against type 2 diabetes was assayed according to method of Sauma L. et al. [31]. HepG2 cells were transfected with plasmid pppre-luc and plasmid phrl-tk. pppre-luc was a plasmid in which the PPAR-responsive element (PPRE) and the firefly luciferase reporter gene were transferred, while plasmid phrl-tk as internal reference was transfected with Renilla luciferase reporter gene. After the transfected cells were seeded in 96-well plates at a cell density of /well and cultured in the low-sugar DMEM containing 100 U ml 1 of streptomycin and 100 U ml 1 of penicillin at 37 o C overnight. The medium was refreshed with low-sugar DMEM containing the sample under testing. The blank control (non-transfected cells), negative control (cells transfected and added no samples), and positive control (cells transfected and added pioglitazone) were included and cultured under the same conditions. After an additional 24-h culture, the luciferase activity and excitation rate were determined by measuring chemiluminescence intensity L using a Dual- Luciferase Reporter Gene Assay Kit (Promega, USA). L1 represented the firefly luciferase chemiluminescence intensity, while L2 represented the L value of internal reference. The excitation rate (ER) was calculated using the following equation: (L1 sample L1 blank ) / (L1negative L1 blank ) ΕR 100% (L2 L2 )/(L2 L2 ) sample blank negative blank The anti-diabetic activity was considered as extremely high when excitation-rate value was greater than 100%. All samples were analyzed twice at concentration of 10 μg ml 1. Screening for antibiotics biosynthetic genes Antibiotic biosynthesis potential of the 80 strains was estimated by PCR screening. Five related genes encoding key enzymes were tested, including Type I and type II (aromatic) polyketide synthetases (PKS-I & PKS-II), nonribosomal peptide synthetase (NRPS), 3-amino-5-hydroxybenzoic acid synthase (AHBA), and glycopeptides associated gene oxyb. Five sets of PCR primers were used: K1F (5 -TSA AGT CSA ACA TCG GBC A-3 ) and M6R (5 -CGC AGG TTS CSG TAC CAG TA-3 ) targeting PKS-I sequences [16] ; A3F (5 -GCS TAC SYS ATS TAC ACS TCS GG-3 ) and A7R (5 -SAS GTC VCC SGT SCG GTA S-3 ) targeting NRPS sequences [16] ; ARO-PKS-F (5 -GGC AGC GGI TTC GGC GGI TTC CAG-3 ) and ARO-PKS-R (5 -CGI TGT TIA CIG CGT AGA ACC AGG CG-3 ) targeting PKS-II sequences [32] ; ANSA-F (5 -CCS GCS TTC ACS TTC ATC TC-3 ) and ANSA-R (5 -AIS YGG AIC ATI GCC ATG TAG-3 ) targeting 3, 5-AHBA synthase gene rifk sequences [32] ; rifk gene of Amycolatopsis mediterranei was used as positive control; Foxy (5 -CTG GTC GGC AAC CTG ATG GAC-3 ) and Roxy (5 -CAG GTA CCG GAT CAG CTC GTC-3 ) targeting P450 mono-oxygenase gene oxyb associated with glycopeptides antibiotics [32]. The amplify system was similar to the amplify system of 16S rrna except for an addition of 2.5 μl DMSO per tube [33]. PCR reaction procedure was performed as follows: (1) 5 min pre-denaturation at 96 o C; (2) 35 cycles of 45-s denaturation at 96 o C, 30-s annealing at 55 o C (for PKS-I), 59 o C (for NRPS), 64 o C (for PKS-II), 56 o C (for ANSA) and 64 o C (for oxyb), and 45-s extension at 72 o C; and (3) 10 min final extension step at 72 o C. Aliquots of the PCR products (5 μl) were visualized by 1% agarose gel electrophoresis stained with ethidium bromide. Nucleotide sequence accession numbers The sequences reported in the present study have been deposited in the GeneBank database. The accession numbers are HQ HQ000036, HM HM149231, JF JF261626, and JQ JQ (Table 1). Statistical analysis Statistical calculations were made using R project Each sample was tested in duplicate and the test was repeated twice. The data are presented as means standard deviation. We used Student s t-test to determine the statistical significance. P 0.05 was considered statistically significance. Results Identification and phylogenetic analysis of representative isolates Based on cultural and morphological characteristics, 80 out of 119 actinomycetes isolated were selected for molecular identification. Their host sources and accession numbers are shown in Table 1. The 16S rrna gene partial sequence (610 bp) analysis indicated that 66 strains shared 98% 100% of 16S rdna similarities with Streptomyces spp. The other 14 strains were consisted of 2 Amycolatopsis, 4 Actinomadura, 1 Nocardia, 2 Microtetraspora, 3 Micromonospora, 1 Kribbella, and 1 Lentzea, indicating a great taxonomical diversity of endophytic actinomycetes in these plants. Phylogenetic analysis of the strains from the 13 medicinal plants was investigated and the reustls are presented in Fig. 1. Based on 16S rdna sequences, phylogenetic trees of the 35 strains from Dioscorea bulbifera, Chenopodium album, and Populus adenopoda were separately constructed and analyzed in the present study. Other isolates from Podocarpus 946

6 macrophyllus, Forsythia suspensa, and Solanum torvum have been published in previous reports [34-35]. 16 strains isolated from D. bulbifera were sorted to seven different clades (Fig. 2). 11 strains isolated from C. album and eight strains from P. adenopoda were both sorted to six different clades (Figs. 3 and 4), with 8 isolates from P. adenopoda being affiliated to 4 genera. All the three groups showed considerable high diversity among the medicinal plants. These results suggested that actinomycetes inhabited in the unique niches inside medicinal plant tissues with abundant diversity of genotypes. Streptomyces was the most frequently isolated genus from these plants, and rare actinomycetes were demonstrated to be able to dwell interiorly too. Antimicrobial activity of endophytic actinomycetes The samples of fermentation broth of the arid crude extracts of strains were used for antimicrobial activity screening against ten pathogenic indicator microorganisms. Each sample was tested in duplicate to calculate the average values of inhibition-zone diameters. 12 (15%) out of the 80 strains showed antimicrobial activity against at least one of indicator microorganisms, which were all affiliated to the genus Streptomyces. 947

7 Fig. 1 Neighbour-joining phylogenetic tree of 80 endophytic actinomycetes strains isolated from 13 traditional medicinal plants in Sichuan province, based on the partial 16S rrna gene sequences. CR: Celastrus rugosus Rehd. et Wils; DB: Dioscorea bulbifera Linn.; CA: Chenopodium album Linn.; PA: Populus adenopoda Maxim; FS: Forsythia suspensa (Thunb.) Vahl; AC: Aconitum carmichaeli Debx; PM: Podocarpus macrophyllus (Thunb.) Sweet; RD: Rumex dentatus Linn.; PR: Perrottetia racemosa (Oliv.) Loes; ST: Solanum torvum Swartz; GC: Geranium carolinianum Linn.; CC: Cinnamomum camphora (Linn.) Presl; IN: Impatiens nolitangere Linn. Fig. 2 Neighbour-joining phylogenetic tree based on the partial 16S rrna sequences showing the affiliation of representatives from Dioscorea bulbifera. GeneBank accession numbers of the isolates and type strains are given in the parentheses. The numbers at the branching nodes are the percentages of occurrence in replicated bootstrapped trees, 5 nt substitution per 100 nt. antimicrobial activity; anticancer activity; anti-diabetes activity; positive occurrence of 2 genes; positive occurrence of 3 genes; positive occurrence of 4 genes 948

8 Fig. 3 Neighbour-joining phylogenetic tree based on the partial 16S rrna sequences showing the affiliation of representatives from Chenopodium album. GeneBank accession numbers of the isolates and type strains are given in the parentheses. The numbers at the branching nodes are the percentages of occurrence in replicated bootstrapped trees, 2 nt substitution per 100 nt. antimicrobial activity; anticancer activity; anti-diabetes activity; positive occurrence of 2 genes; positive occurrence of 3 genes; positive occurrence of 4 genes Fig. 4 Neighbour-joining phylogenetic tree based on the partial 16S rrna sequences showing the affiliation of representatives from Populus adenopoda. GeneBank accession numbers of the isolates and type strains are given in the parentheses. The numbers at the branching nodes are the percentages of occurrence in replicated bootstrapped trees, 5 nt substitution per 100 nt. antimicrobial activity; anticancer activity; anti-diabetes activity; positive occurrence of 2 genes; positive occurrence of 3 genes 949

9 The members of rare actinomycetes were generally limited in their antimicrobial activity. On the other hand, varying antibacterial activity was observed for the isolates; 4 strains displayed acute and wide antibacterial spectrum of more than 4 indicator pathogens (Table 2). Strains A229, A243, and A281 showed comparatively strong (Φ 15 mm) and wide antibacterial spectrum. Furthermore, the active isolates showed certain selectivity to some pathogens. The activity against K. pneumoniae was most frequently observed (83.3%), with relatively high proportion of activities against B. subtilis and S. aureus being 66.7% and 50.0%, respectively. In contrast, all test strains showed no inhibition against 4 pathogenic fungi and E. coli, except for the strain A301 which displayed moderate inhibition against A. fumigatus (data not showed). Anticancer and anti-diabetic activities of endophytic actinomycetes Our activity screening results revealed that the metabolites of 80 isolates had prominent anticancer and anti-diabetic activities (Tables 2 and 4), with 87.5% and 58.8% of the isolates showing anticancer and anti-diabetic activities, respectively. As an initial screening for anticancer activity, we only investigated the in vitro anticancer activity against HepG2 cells in the present study. 26 strains (32.5%) displayed significant anticancer activity. Ten strains, including A280, A270, A263, A279, A243, A221, A229, A276, A281, and A290, were be considered as prominent candidates for further research. Most of them were Streptomyces, except for strain A290 which was assigned to Micromonospora. In contrast, 25 strains (31.3%) showed moderate (GR: 70 80) and 19 strains (23.8%) showed weak (GR: 80 90) inhibitory activity against HepG2 cells, including several rare actinomycetes, such as strains A245, A246, A248, A235, and A223. They were affiliated to Actinomadura, Nocardia, Microtetras pora, Actinocorallia, and Amycolatopsis, respectively. Table 3 Partial results of PCR screening for antibiotic synthesis genes Positive results of PCR screening Strain no. Genus Host PKS-I PKS-II NRPS ANSA oxyb A211 Streptomyces sp. Perrottetia racemosa A217 Streptomyces sp. Dioscorea bulbifera A219 Amycolatopsis sp. Dioscorea bulbifera A221 Streptomyces sp. Dioscorea bulbifera A229 Streptomyces sp. Dioscorea bulbifera A236 Streptomyces sp. Chenopodium album A238 Streptomyces sp. Chenopodium album A239 Streptomyces sp. Chenopodium album A241 Streptomyces sp. Podocarpus macrophyllus A243 Streptomyces sp. Podocarpus macrophyllus A245 Actinomadura sp. Podocarpus macrophyllus + + A246 Nocardia sp. Populus adenopoda - + A248 Microtetraspora sp. Populus adenopoda + + A251 Actinocorallia sp. Populus adenopoda + + A252 Microtetraspora sp. Populus adenopoda + + A255 Streptomyces sp. Forsythia suspensa A260 Streptomyces sp. Forsythia suspensa A263 Streptomyces sp. Forsythia suspensa A267 Streptomyces sp. Rumex dentatus A270 Streptomyces sp. Cinnamomum camphora A274 Streptomyces sp. Solanum torvum A275 Kribbella sp. Solanum torvum + + A289 Micromonospora sp. Impatiens nolitangere A291 Micromonospora sp. Impatiens nolitangere + + A301 Streptomyces sp. Geranium carolinianum A318 Streptomyces sp. Aconitum carmichaeli A322 Lentzea sp. Aconitum carmichaeli A326 Streptomyces sp. Chenopodium album A327 Streptomyces sp. Chenopodium album A329 Streptomyces sp. Chenopodium album

10 Table 4 Anticancer andanti-diabetiv activities and positive PCR screening for selected genes Taxa group Anticancer Anti-diabetes PKS-I PKS-II NRPS ANSA oxyb PG (> 3) PG (> 4) RA 1 (7.1) 4 (28.6) 7 (50.0) 4 (28.6) 13 (92.9) 4 (28.6) 1 (7.1) 4 (28.6) 1 (7.1) Streptomyces 25 (37.9) 13 (19.7) 37 (56.1) 43 (65.2) 59 (89.4) 11 (16.7) 6 (9.1) 33 (50.0) 7 (10.6) Total 26 (32.5) 17 (21.3) 44 (55.0) 47 (58.8) 72 (90.0) 15 (18.8) 7 (8.8) 37 (46.3) 8 (10.0) Note: RA represents rare actinomycetes; PG represents sum of positive genes; numbers in the parentheses were relevant percentage of positive-strain sum; the percentage is respectively related to positive occurrence rate of rare actinomycetes or Streptomyces As shown in Tables 2 and 4, 17 strains (21.3%) displayed significant anti-diabetic activity with the values of excitation rate (ER) being over 50; among them, the strains A236, A224, A238, A245, and A235 showed remarkable ER values of over 125. In addition, 12 strains (15.0%) and 17 strains (21.3%) showed moderate (ER: 30 50) and weak (ER: 10 30) anti-diabetic activities, respectively. Furthermore, there was a difference in strain distribution pattern for the two types of activities among the strains., The prominent candidates for anticancer activity presented limited proportion of rare actinomycetes (7.1%), while for anti-diabetic activity the candidates had a high percentage (28.6%) of rare actinomycetes (Table 4). Detection of antibiotic biosynthetic genes The antibiotic biosynthesis potentials of the 80 representative isolates were investigated by specific amplification. In order to detect the potential antibiotic biosynthetic genes, we selected five genes involved in the production of widespread antibiotics. For example, PKS-I and PKS-II are involved in the production of Type-I polyketides and Type-II polyketides, respectively; NPRS is involved in the production of nonribosomal peptides; ANSA is involved in the production of ansamycins; and oxyb is involved in the production of glycopeptide antibiotics [16, 32]. In the present study, 78 out of the 80 strains possessed at least one of the test genes. The positive amplification rates of five genes PKS-I, PKS-II, NRPS, ANSA (rifk), and oxyb were 55.0%, 58.8%, 90.0%, 18.8% and 8.8%, respectively (Tables 3 and 4). On the other hand, the detection rates of the five genes varied between members of Streptomyces and other genera. NRPS genes displayed the highest positive rate in the members of both Streptomyces and rare actinomycetes (89.4% and 92.9%, respectively). The positive rate of PKS-II genes was comparatively higher in Streptomyces (65.2%), while that of ANSA genes was higher in rare actinomycetes (28.6%, Table 4). However, this differentiation of taxa was not distinctive for PKS-I and oxyb genes. Furthermore, 46.3% of the isolates presented positive results of more than three types of biosynthesis genes, in which strains A217, A239, A241, A255, A263, A289, A326, and A327 possessed more than four types of biosynthesis genes. Discussion Historically, actinomycetes have been the origin of the largest number of new antibiotic drug candidates and lead compounds with applications in many other therapeutic areas [2], and endophytic actinomycetes are now considered as exciting novel sources for obtaining new bioactive compounds [11, 20, 36-37]. In the present study, we investigated endophytic actinomycetes associated with traditional medicinal plants from endemic forest of Qingcheng and Emei Mountains in Sichuan. Our results indicated many traditional medicinal plants possess a great diversity in populations of endophytic actinomycetes community (Figs. 1 4). Streptomyces (66 out of 80 strains) manifested a dominant status among all the test plants, which has also been noted by other groups [11, 18, 20, 38]. The diversity of endophytes was related to the selection of host plants and isolation of tissues [39]. The tissue samples of D. bulbifera and C. album were sprouted bulbuls and roots, respectively, whereas the stems from P. adenopoda were aerial tissues. Therefore, the endophytic actinomycetes communities of D. bulbifera and C. album revealed greater diversity than P. adenopoda. It has been reported that endophytes from the perennial plants have a greater diversity than those from seasonal herbs [4, 12]. Similarly, our data indicated that the taxa affiliation of actinomycetes from P. adenopoda manifested a relative abundant population of rare actinomycetes. These results suggested actinomycetes inhabited interior of medicinal plants tissues might have abundant diversity and host preference. It is believed that plant endophytes provide protection for hosts by producing a variety of bioactive metabolites [40]. Many endophytic actinobacteria inhibit or kill a wide variety of pathogenic microorganisms [41-43]. In the present study, after multiple activity screening among the isolates, many prominent candidates for further exploitation were identified. Among them, 12 strains showed antimicrobial activity, with strains A229, A243 and A281 showing acute and wide antibacterial spectra (Table 2). 87.5% and 58.8% of the isolates showed anticancer and anti-diabetic activities, respectively, in which 10 strains displayed strong anticancer activity and 5 strains displayed significant anti-diabetic activity (Table 2). Among potential candidates with antimicrobial and anticancer activities, Streptomyces was the dominant genus. However, rare actinomycetes of good anti-diabetic activity occupied a relatively high ratio (28.6%) than those of anticancer (7.1%, Table 4). Several researches have shown that endophytes can protect host plants by producing antimicrobial compounds [4]. Some of these compounds possess antifungal, antibacterial, and antimalarial activity, among other bioactivities, especially for the endophytes from medicinal plants producing useful antibiotics [4, 42]. The activity types of the strains shared similarity with that of relative host plants [42, 44]. D. bulbifera is a toxic perennial ethnomedical herb used for detoxification and relieving cough and asthma [45]. D. bulbifera produces a series 951

11 of unique and toxic compounds diosbulbins [45]. C. album is an annual herbaceous plant with slight toxicity andused as a traditional antibacterial medicine for curing dysentery and diarrhea [46]. In the present study, the isolates from D. bulbifera revealed greater anticancer activity, compared with those from C. album. The latter showed minimal cytotoxity but high anti-diabetic activity (Table 2, Figs. 2 and 3). P. adenopoda is a perennial ligneous ethnomedical plant with virtues of alleviating pain and promoting blood circulation [47]. Considering that the host was nontoxic, its isolates were observed with no clear antimicrobial or anticancer activities (Fig. 4). As concluded in their reviews by Rosenblueth et al. [44] and Sachiko H et al. [40], the plants seem to have intensive interactions with a variety of endophytes in nature. The genomic approaches aiming at exploring the biosynthetic potential of actinomycetes have achieved extraordinary development by many research groups [16, 37, 48]. Our results from the present study indicated that the detection rates of PKS-I, PKS-II, NRPS, ANSA and glycopeptides (oxyb) in these strains were 55.0%, 58.8%, 90.0%, 18.8% and 8.8%, respectively (Tables 3 and 4). The higher proportions of PKS-I, PKS-II, NRPS and lower detection rates of ANSA were in agreement with that of many previous researches of actinomycetes obtained from other environments [16, 18, 32, 46]. NRPS genes showed wide distribution in the members of both Streptomyces and rare actinomycetes (89.4% and 92.9%). Rare actinomycetes manifested comparatively prominent potential of anti-diabetic activity and relative higher occurrence of ANSA genes. However, Streptomyces displayed significant antimicrobial and anticancer activities and relative higher distribution of PKS-II genes (Table 4). Nevertheless, the occurrence of multiple biosynthesis genes in some of the isolates could not be directly related to the production of activities. For example, the strains with multiple antibiotic biosynthesis pathway (PG > 4) (Tables 2 and 4), such as strains A217, A263, and A289, revealed anticancer and antibacterial activities, whereas strains A239, A241, and A255 displayed no such activities. The reported results of PCR screening for genes of antibiotics biosynthesis have demonstrated that there are many potential biosynthesis pathways of antibiotics [16, 49-50]. Our results from the present study also supported the notion that the gene mining approach is conducive to find potential secondary metabolites. In conclusion, our results from the present study demonstrated that endophytic actinomycetes from traditional medicinal plants would be reliable source for natural bioactive compounds screening. Our PCR screening results revealed a rich metabolic potential of the isolates. Considering that endophytic Streptomyces from various hosts have been found to produce prolific novel antibiotics and many other compounds with impressive activities [14-15, 41-42], our observations from the present study further support the notion that endophytic actinomycetes have the ability to produce a wide range of antibacterial, antifungal, and biological control agents, and plant growth regulatory metabolites, offering various applications as novel pharmaceutical and agricultural agents. Acknowledgements We thank the Institute of Materia Medica of Chengdu Diao pharmaceutical Corp. for conducing the anticancer and anti-diabetic activity screening and Microbial Resources Center (MRC) of Sichuan Industrial Institute of Antibiotics (SIIA) for providing indicator pathogens. References [1] Demain AL, Sanchez S. Microbial drug discovery: 80 years of progress [J]. J Antibiot, 2009, 62(1): [2] Bérdy J. Bioactive microbial metabolites [J]. 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