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Research ArticleEndophytic Actinobacteria Associated withDracaena cochinchinensis Lour Isolation Diversityand Their Cytotoxic Activities

Nimaichand Salam1 Thi-Nhan Khieu12 Min-Jiao Liu3 Thu-Trang Vu2 Son Chu-Ky2

Ngoc-Tung Quach4 Quyet-Tien Phi4 Manik Prabhu Narsing Rao1 Angeacutelique Fontana5

Samira Sarter5 andWen-Jun Li13

1State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources School of Life SciencesSun Yat-Sen University Guangzhou 510275 China2Department of Food Technology School of Biotechnology and Food Technology Hanoi University of Science and TechnologyHanoi Vietnam3Yunnan Institute of Microbiology Yunnan University Kunming 650091 China4Laboratory of Fermentation Technology Institute of Biotechnology Vietnam Academy of Science and Technology Hanoi Vietnam5CIRAD UMR QUALISUD 34398 Montpellier France

Correspondence should be addressed to Son Chu-Ky sonchukyhusteduvn and Wen-Jun Li liacthotmailcom

Received 30 June 2016 Accepted 20 March 2017 Published 6 April 2017

Academic Editor Young-Mi Lee

Copyright copy 2017 Nimaichand Salam et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Dracaena cochinchinensis Lour is an ethnomedicinally important plant used in traditional Chinese medicine known as dragonrsquosblood Excessive utilization of the plant for extraction of dragonrsquos blood had resulted in the destruction of the important nicheDuring a study to provide a sustainable way of utilizing the resources the endophytic Actinobacteria associated with the plantwere explored for potential utilization of their medicinal properties Three hundred and four endophytic Actinobacteria belongingto the genera StreptomycesNocardiopsis BrevibacteriumMicrobacterium Tsukamurella Arthrobacter BrachybacteriumNocardiaRhodococcus Kocuria Nocardioides and Pseudonocardia were isolated from different tissues of D cochinchinensis Lour Of these17 strains having antimicrobial and anthracyclines-producing activities were further selected for screening of antifungal andcytotoxic activities against two human cancer cell lines MCF-7 and Hep G2 Ten of these selected endophytic Actinobacteriashowed antifungal activities against at least one of the fungal pathogens of which three strains exhibited cytotoxic activities withIC50-values ranging between 3 and 33120583gsdotmLminus1 Frequencies for the presence of biosynthetic genes polyketide synthase- (PKS-) IPKS-II and nonribosomal peptide synthetase (NRPS) among these 17 selected bioactive Actinobacteria were 294 706 and235 respectively The results indicated that the medicinal plantD cochinchinensis Lour is a good niche of biologically importantmetabolites-producing Actinobacteria

1 Introduction

Actinobacteria especially the genus Streptomyces are majorproducers of bioactive metabolites [1] and account for nearly75 of the total antibiotic production available commercially[2 3] A few decades ago antibiotics were considered aswonder drugs since theywarded off deadly pathogens leading

to eradication of infectious diseases However the unprece-dented deployment of antibiotics over a period of time hasresulted in evolution of multidrug-resistant pathogensThereis increasing attention to bioprospecting of Actinobacteriafrom different biotopes With limiting bioresources it isnow imperative for search of unexplored or underexploredhabitats One such overlooked and promising niche is the

HindawiBioMed Research InternationalVolume 2017 Article ID 1308563 11 pageshttpsdoiorg10115520171308563

2 BioMed Research International

inner tissues of plants especially those with ethnomedicinalvalue [4ndash10]

TheplantDracaena cochinchinensisLour has beenused asa traditional folkmedicine in the oriental countries includingChina [11] D cochinchinensis Lour has many medicinallyimportant properties like antimicrobial antiviral antitumorcytotoxic analgesic antioxidant anti-inflammatory haemo-static antidiuretic antiulcer andwoundhealing activities [1012]The plant is the source of deep red resin havingmedicinalproperties which is also known as dragonrsquos blood The maincomponents of dragonrsquos blood are flavonoids and stilbenoids[13] Apart from its medicinal use it also finds applicationsas colouring materials and wood varnish [12] The slowgrowth of the plant along with low yield of dragonrsquos bloodextracts however led to the destruction of large numberof these plants thereby endangering the plant The currentstudy described the diversity of culturable Actinobacteriaassociated with this medicinal plant and also indicated thecytotoxic potential of these Actinobacteria The study ina way proposed a means for sustainable use of the plantresources without destroying the natural niche

2 Materials and Methods

21 Sample Collection and Isolation of Endophytic Actinobac-teria Healthy plant samples (leaves stems and roots) ofmedicinal plant D cochinchinensis Lour were collected fromfour different provinces located in two countries Pingx-iang Guangxi province China (20∘0610158400210158401015840N 106∘4510158400110158401015840Eelevation 236m) Xishuangbanna Yunnan province China(21∘5510158404110158401015840N 101∘2510158404910158401015840E 984m) Bach Ma National ParkThuaThien Hue province Vietnam (16∘910158405510158401015840N 107∘5510158401910158401015840E1450m) andCuc PhuongNational Park Ninh Binh provinceVietnam (20∘191015840810158401015840N 105∘3710158402010158401015840E 338m)The plant sampleswere packed in sterile plastics taken to the laboratory andsubjected to isolation procedures within 96 h The sampleswere washed thoroughly with running tap water and inultrasonic bath to remove any adhering soil particles and air-dried at ambient temperature for 48 h

Two methods were employed for the isolation of theendophytic Actinobacteria using seven specific isolationmedia (Table 1)

Method 1 The plant parts of D cochinchinensis Lour wereexcised and subjected to a five-step surface-sterilizationprocedure a 4min wash in 5 NaOCl followed by 10minwash in 25 Na2S2O3 a 5min wash in 75 ethanol a washin sterile water and a final rinse in 10 NaHCO3 for 10minAfter drying thoroughly under sterile conditions the surfacesterilized tissues were disrupted aseptically in a commercialblender and distributed on isolation media [5 7]

Method 2 The surface sterilized plant parts (1-2 g) weresliced groundedwithmortar and pestle andmixedwith 05 gCaCO3 The samples were kept in a laminar flow cabinet for14 d incubated at 80∘C for 30min and plated onto isolationmedia [7]

Each medium was supplemented with nalidixic acid(25mgsdotLminus1) nystatin (50mgsdotLminus1) and K2Cr2O7 (50mgsdotLminus1)to inhibit the growth of Gram-negative bacteria and fungipolyvinyl pyrrolidone (2) and tannase (0005) were alsoadded to improve the development of colonies on mediaColonies grown on these isolation media were selected andpurified by repeated streaking on YIM 38 medium The purecultures were preserved as glycerol suspensions (20 vv)at minus80∘C and as lyophilized spore suspensions in skim milk(15 wv) at 4∘C

22 Identification and Diversity Profiling For phyloge-netic characterization genomics DNAs of all isolates wereextracted using an enzyme hydrolysis method About 50mgof the freshly grown culture was taken in an autoclaved15mL Eppendorf tube To the culture 480 120583L TE buffer (1x)and 20 120583L lysozyme solution (2mgsdotmLminus1) were added Thebacterial suspensionwas thoroughlymixed and incubated for2 h under shaking conditions (160 rpm 37∘C) The mixturewas treated with 50120583L SDS solution (20 wv) and 5 120583LProteinase K solution (20 120583gsdotmLminus1) and kept on a waterbath (55∘C 1 h) DNA was then extracted twice with phenol-chloroform-isoamyl alcohol (25 24 1 vvv) followed byprecipitation with 80 120583L sodium acetate (3molsdotLminus1 pH48ndash52) and 800120583L absolute ethanol The resulting DNAprecipitate was centrifuged at 4∘C (12000 rpm 10min)washed with 70 ethanol and then air-dried The extractedDNA was resuspended in 30 120583L TE buffer and storedat minus20∘C PCR amplification for 16S rRNA gene fromthe extracted DNA samples was done using the primerpair PA-PB (PA 51015840-CAGAGTTTGATCCTGGCT-31015840 PB 51015840-AGGAGGTGATCCAGCCGCA-31015840) as described previously[14] Amplified PCR products were purified and sequencedby Sangon Biotech (Shanghai) Identification of phyloge-netic neighbours and calculation of pairwise 16S rRNAgene sequence similarities were achieved using the EzTaxonserver (httpwwweztaxonorg) [15] and BLAST analysis(httpblastncbinlmnihgovBlastcgi) The alignment ofthe sequences was done using CLUSTALW [16]The phyloge-netic tree was constructed using the aligned sequences by theneighbour-joining method [17] using Kimura 2-parameterdistances [18] in the MEGA 6 software [19] To determine thesupport of each clade bootstrap analysis was performed with1000 replications [20]

23 Selection of Bioactive Actinobacteria Strains Each of theisolated Actinobacteria was screened for antimicrobial activ-ity and anthracyclines production The antibacterial activi-ties were evaluated against Methicillin-resistant Staphylococ-cus epidermidis (MRSE) ATCC 35984 Methicillin-resistantStaphylococcus aureus (MRSA) ATCC 25923 Methicillin-susceptible Staphylococcus aureus (MSSA) ATCC 29213Klebsiella pneumoniae ATCC 13883 Aeromonas hydrophilaATCC 7966 and Escherichia coli ATCC 25922 using theagar well diffusion method [21] Anthracycline productivitywas screened using the pigment production test as describedby Trease [22] Based on the results of the two screeningsbioactive strains were selected for further assays

BioMed Research International 3

Table 1 Composition of the seven media used for the isolation of endophytic Actinobacteria from Dracaena cochinchinensis Lour

Medium Name and composition (g Lminus1of water) Reference

1 Tap water-yeast extract agar (TWYE)Yeast extract 025 K2HPO4 05 agar 15

[3 5]

2 Trehalose agarTrehalose 6 KNO3 05 CaCl2 03 Na2HPO4 03 MgSO4sdot7H2 O 02 agar 15 [5]

3Sodium propionate agarSodium propionate 2 NH4NO3 01 KCl 01 MgSO4sdot7H2 O 005 FeSO4sdot7H2 O 005agar 15

[5]

4Starch agarStarch 2 KNO3 1 NaCl 04 K2HPO4 05 MgSO4sdot7H2 O 05 FeSO4sdot7H2 O 001 agar15

[5]

5Citrate agarCitric acid 012 ferric ammonium citrate 012 NaNO3 15 K2 HPO4sdot3H2 O 04MgSO4sdot7H2 O 01 CaCl2sdotH2O 005 EDTA 002 Na2CO3 02 agar 15

This study

6Sodium propionate-asparagine-salt agarSodium propionate 4 asparagine 1 casein 2 K2HPO4 1 MgSO4sdot7H2 O 01FeSO4sdot7H2 O 001 NaCl 30 agar 15

[5]

7Dulcitol-proline agarDulcitol 2 proline 05 K2HPO4 03 NaCl 03 MgSO4sdot7H2 O 1 CaCl2sdot2H2 O 1 agar15

This study

24 Antifungal and Cytotoxicity Tests Antifungal activity ofthe selected bioactive strains was tested against Fusariumgraminearum Aspergillus carbonarius and Aspergillus west-erdijkiae (strains producing the mycotoxins deoxynivalenoland ochratoxin A) [23 24] These test pathogens were pro-vided by CIRAD UMRQUALISUD France and maintainedon Potato Dextrose Agar (PDA)

The cytotoxic activity of the selected strains was testedby sulforhodamine B (SRB) assay as described earlier [25ndash27]The human breast adenocarcinoma (MCF-7) and humanhepatocellular carcinoma (Hep G2) cells lines used for thetest were procured from American Type Culture Collection(ATCC BoulevardManassa VA 20110 USA) Ellipticine wasused as the positive control

25 Screening for Biosynthetic Genes Three sets of PCRprimers A3FA7R K1FM6R and KS120572FKS120572R were used foramplification of nonribosomal peptide synthetase (NRPS)polyketide synthase- (PKS-) I and PKS-II specific domains[6 28] PCR amplifications were performed in a Biome-tra thermal cycler in a final volume of 25120583L containing02 120583molsdotLminus1 of each primer 01 120583molsdotLminus1 of each of the fourdNTPs (Takara Japan) 25 120583L of extracted DNA 05 unitof Taq DNA polymerase (with its recommended reactionbuffer) and 10 of DMSO Amplifications were performedaccording to the following profile initial denaturation at96∘C for 5min 30 cycles of denaturation at 96∘C for 1minprimer annealing at either 57∘C (for K1FM6R A3FA7R) or58∘C (for KS120572FKS120572R) for 1min and extension at 72∘C for1min followed by a final extension at 72∘C for 5min Thesizes of amplicons were 1200ndash1400 bp (K1FM6R) 613 bp(KS120572FKS120572R) and 700ndash800 bp (A3FA7R)

3 Results

31 Isolation of Endophytic Actinobacteria A total of 304putative endophytic Actinobacteria were isolated from threedifferent tissues of D cochinchinensis Lour The highestnumber ofActinobacteriawas isolated from roots (117 strains3849) followed by stems (113 strains 3717) and leaves(74 strains 2434) (Figure 1) Among the sites moreActinobacteria were isolated from Xishuangbanna (Yunnanprovince China) and Cuc Phuong National Park (Ninh Binhprovince Vietnam) (Figure 1)

During the present study Method 2 was found to bemore suitable for the isolation of endophytic Actinobacteriafrom tissues of D cochinchinensis Lour and accounted fornearly 65 of the total isolation All the media used in thecurrent study except for sodium propionate-asparagine-saltagar were suitable for isolation of endophytic Actinobacteria(Figure 2)

32 Diversity Profiling Based on the 16S rRNAgene sequenceanalysis the most abundant Actinobacteria genera wereStreptomyces (8684) followed by Nocardiopsis (493)Brevibacterium (164) Microbacterium (164) Tsuka-murella (164) Arthrobacter (066) Brachybacterium(066) Nocardia (066) Rhodococcus (066) Kocuria(033) Nocardioides (033) and Pseudonocardia (033)The relative abundance of the endophytic Actinobacteriaamong the different sites is shown in Table 2 Among thedifferent sampling sites Yunnan and Ninh Binh yielded thehighest diversity each contributing eight genera of Acti-nobacteria Yunnan samples yielded the genera Streptomyces


14

18

15

318

12 13

52

3374

113

117

Tota

l

Nin

h Bi

nh

Gua

ngxi

Yunn

an

LeavesStemsRoots

48

44

34

ua

ie

n H

ue

Figure 1 Distribution of endophytic Actinobacteria isolated fromthe different tissues of Dracaena cochinchinensis Lour among thedifferent sampling sites

Medium 1Medium 2

Medium 3

Medium 4

Medium 5

Medium 6

Medium 7

3

18

18

19

16

1115

Figure 2 Effect of media on the isolation of endophytic Actinobac-teria

Nocardiopsis Brevibacterium Microbacterium Brachybac-terium Rhodococcus Kocuria and Tsukamurella while NinhBinh samples yielded Streptomyces TsukamurellaNocardiop-sisArthrobacterNocardiaBrevibacteriumNocardioides andPseudonocardia Thua Thien Hue samples contained Strepto-myces Nocardiopsis andMicrobacterium while Streptomycesand Nocardiopsis were present in Guangxi samples

33 Selection of Bioactive Actinobacteria Strains All 304Actinobacteria isolates were tested for antimicrobial activityand anthracycline production Table 3 represents the distri-bution of bioactive Actinobacteria These bioactive strainswere distributed in the genera Streptomyces NocardiopsisNocardioides Pseudonocardia and Tsukamurella The genusStreptomyces possessed the highest proportion of isolateswith antimicrobial activities Anthracyclines are importantgroup of antitumor antibiotics and are being used in cancertreatment [29 30] Of the 304 strains 49 strains testedpositive for anthracycline production

Based on the results of the bioactivity screening 17strains (HUST001-HUST011 HUST013-HUST015 HUST017HUST018 and HUST026) were selected for further antifun-gal and cytotoxicity studies (Table 4) Of the 17 strains 14belonged to the genera Streptomyceswhile the rest comprisedNocardioides Nocardiopsis and Pseudonocardia (Figure 3)

34 Evaluation of Antifungal and Cytotoxicity Effects of theBioactive Strains Several strains among the selected bioac-tive Actinobacteria were positive for antifungal activitiesagainst the mycotoxins-producing F graminearum A car-bonarius and A westerdijkiae strains Frequencies of theantifungal activities against the indicator fungal pathogenswere as follows F graminearum 588 A carbonarius412 and A westerdijkiae 235 Table 5 summarizes theantifungal profile of the selected 17 strains

Of the 17 strains three strains (HUST001 HUST004 andHUST005) exhibited cytotoxic effects against the two testedhuman cancer cell lines MCF-7 and Hep G2 (Table 5) StrainHUST004 showed significant inhibition toward MCF-7 cellswith IC50-value of 3 120583gsdotmLminus1 while strains HUST001 andHUST005 showed moderate activity with IC50-values of 19and 25120583gsdotmLminus1 respectively Against Hep G2 cell lines IC50-values for the strains HUST004 and HUST005 were 10 and33 120583gsdotmLminus1 respectively The remaining strains were inactiveagainst the two cancer cell lines

35 Screening of Biosynthetic Genes All 17 bioactive strainswere investigated for the presence of PKS-I PKS-II andNRPS genes Frequencies of positive PCR amplification of thethree biosynthetic systems were 2941 7059 and 2353respectively (Table 5) All these three genes were detectedin two strains (HUST003 HUST004) which were identifiedas members of the genus Streptomyces PKS-II gene wasdetected at highest frequencies in both Streptomyces and non-Streptomycetes genera while PKS-I and NRPS genes weredetected only in the genus Streptomyces

4 Discussion

The plant source D cochinchinensis is known for the pro-duction of dragonrsquos blood [11] Traditional practices of folkmedicine involved extraction of dragonrsquos blood from theplant During its extraction large scale exploitation of theplant is necessary owing to the low yield of plantrsquos extractand slow growth of the plant thereby resulting in destructionof large number of century old plant [13] It is therefore


Table 2 Distribution of endophytic Actinobacteria isolated from the different tissues ofD cochinchinensis Lour among the different samplingsites

Genera YunnanChina

GuangxiChina

ThuaThien HueVietnam

Ninh BinhVietnam Total

Arthrobacter 0 0 0 2 2Brachybacterium 2 0 0 0 2Brevibacterium 4 0 0 1 5Kocuria 1 0 0 0 1Microbacterium 4 0 1 0 5Nocardia 0 0 0 2 2Nocardioides 0 0 0 1 1Nocardiopsis 8 1 2 4 15Pseudonocardia 0 0 0 1 1Rhodococcus 2 0 0 0 2Streptomyces 104 46 30 82 262Tsukamurella 1 0 0 5 6Total 126 47 33 98 304

Table 3 Bioactivity profiles of the endophytic Actinobacteria isolated from D cochinchinensis Lour

Genera Antimicrobial activity Anthracycline productionATCC 35984 ATCC 25923 ATCC 29213 ATCC 13883 ATCC 7966 ATCC 25922

Arthrobacter 0 0 0 0 0 0 0Brachybacterium 0 0 0 0 0 0 0Brevibacterium 0 0 0 0 0 0 0Kocuria 0 0 0 0 0 0 0Microbacterium 0 0 0 0 0 0 0Nocardia 0 0 0 0 0 0 0Nocardioides 0 0 1 1 0 0 1Nocardiopsis 0 3 4 1 0 0 1Pseudonocardia 0 0 1 0 1 0 1Rhodococcus 0 0 0 0 0 0 0Streptomyces 70 68 70 70 96 53 46Tsukamurella 0 0 0 0 1 0 0Total 70 71 76 72 98 53 49Proportion () 2303 2326 2500 2368 3243 1743 1611Note Number indicates number of isolates positive for the particular bioactivityATCC 35984 Methicillin-resistant Staphylococcus epidermidis (MRSE) ATCC 25923 Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 29213Methicillin-susceptible Staphylococcus aureus (MSSA) ATCC 13883Klebsiella pneumoniae ATCC 7966Aeromonas hydrophilaATCC 25922 Escherichia coli

imperative to search for alternative source of the plantrsquosmetabolites to preserve the plant in its natural niche Onesuch means is to study the endophytic microbes associatedwith the plant In an earlier study by Cui et al [35] Dcochinchinensis collected from Beijing China had been usedto study the endophytic fungal diversity The study resultedin the isolation of 49 fungal strains distributed into 18 generaIn another study of endophytic microbe associated with Dcochinchinensis Khieu et al [10] had isolated a Streptomycesstrain producing two potent cytotoxic compounds fromplant samples collected from Cuc Phuong National ParkNinh Binh province Vietnam But neither of these studiesdescribed the diversity profile of the Actinobacteria com-munities living in association with the plant As endophytic

Actinobacteria from medicinal plants have been a majorresearch area in the search of new antibiotic-producingstrains [4 7 8 36ndash39] we have selected the same plantsource for in-depth analysis of Actinobacteria communitystructure The present study resulted in the isolation of 304Actinobacteria strains

Many reports suggested that maximum endophytes wererecovered from roots followed by stems and leaves [9 31ndash34] Similar observation was found during our study wherebymore number of isolates was obtained from roots than fromstems or leaves (Table 6) This may be due to the fact thatrhizospheric regions of the soil have higher concentration ofnutrients A report also suggested that microorganism entersvarious tissues of plant from rhizosphere and switched to


Table 4 Isolation and characterization profile of the 17 selected endophytic Actinobacteria

Strain Samplingsitelowast

Isolationmedium

Isolationmethod Source Accession

number Closest homologs Pairwisesimilarity

HUST001 NB 3 2 Stem KT033860 Streptomyces puniceusNBRC 12811T 1000HUST002 GX 2 1 Stem KP317660 Streptomyces violarus NBRC 13104T 9945HUST003 TTH 5 1 Stem KT033861 Streptomyces cavourensis NBRC 13026T 9970HUST004 YN 3 2 Root KT033862 Streptomyces cavourensis NBRC 13026T 1000HUST005 NB 4 2 Stem KT033863 Streptomyces parvulus NBRC 13193T 9973HUST006 NB 3 2 Stem KT033864 Streptomyces rubiginosohelvolus NBRC 12912T 9972HUST007 YN 5 1 Root KT033865 Streptomyces puniceusNBRC 12811T 1000HUST008 TTH 6 2 Stem KT033866 Streptomyces puniceusNBRC 12811T 9980HUST009 YN 3 2 Stem KT033867 Streptomyces puniceusNBRC 12811T 9866HUST010 YN 2 1 Root KT033868 Streptomyces pluricolorescensNBRC 12808T 1000HUST011 GX 3 1 Root KT033869 Streptomyces parvulus NBRC 12811T 1000HUST013 NB 4 1 Root KT033870 Pseudonocardia carboxidivorans Y8T 1000HUST014 TTH 5 1 Root KT033871 Streptomyces augustmycinicus NBRC 3934T 9985HUST015 TTH 7 2 Stem KT033872 Streptomyces violarus NBRC 13104T 9957

HUST017 YN 2 2 Leaf KT033873 Nocardiopsis dassonvillei subsp albirubida DSM40465T

1000

HUST018 NB 1 2 Root KT033874 Streptomyces graminisoli JR-19T 9945HUST026 NB 1 2 Root KT033859 Nocardioides ganghwensis JC2055T 9826lowastYN Xishuangbanna Yunnan province China GX Pingxiang Guangxi province China TTH Bach Ma National Park ThuaThien Hue province VietnamNB Cuc Phuong National Park Ninh Binh province Vietnam

endophytic lifestyles [40 41] Isolation of more isolates usingthe secondmethodmay be attributed to the enrichment of thesamples with calcium carbonate Qin et al [7] have reportedthat calcium carbonate altered the pH to alkaline conditionswhich favour the growth of Actinobacteria

Among various genera isolated Streptomyces is pre-dominantly present in the plant D cochinchinensis Thefinding is consistent with similar studies of endophyticbacteria [6 9 32 33 36] In the present study rare Acti-nobacteria of the genera Arthrobacter Brevibacterium Kocu-ria Microbacterium Nocardia Nocardioides NocardiopsisPseudonocardia Rhodococcus and Tsukamurella were alsoisolated Though Arthrobacter Brevibacterium Microbac-terium Nocardia Nocardioides Nocardiopsis Pseudonocar-dia Rhodococcus and Tsukamurella have been reported asendophytic Actinobacteria of medicinal plant [6 7 31ndash34] this study forms the first report for the isolation ofBrachybacterium and Kocuria (Table 6)

Endophytic Actinobacteria are often associated withantimicrobial properties [6 7 31] This is shown by thehigh proportion of antibacterial activities by endophyticActi-nobacteria associated with D cochinchinensis Lour 2303against ATCC 35984 2326 against ATCC 25923 25against ATCC 29213 2368 against ATCC 13883 3243against ATCC 7966 and 1743 against ATCC 25922 Basedon the preliminary bioactivity profile a set of 17 Actinobac-teria were further studied for antifungal and cytotoxic prop-erties Of the 17 strains selected 10 strains were significantagainst F graminearum seven against A carbonarius and

four against A westerdijkiae Similar findings have beenreported in related studies of Streptomyces strains [42ndash44] Four strains (HUST003 HUST004 HUST005 andHUST026) showed remarkable antifungal activity against alltest fungi (Table 5) In contrast to above strains HUST002HUST006 HUST008 HUST009 HUST013 HUST015 andHUST017 did not show any antifungal activity

In the study of Cui et al [35] it was indicated that 71 ofthe fungal isolates obtained fromD cochinchinensis exhibitedvaried antitumor activities against five human cancer celllines HepG2 MCF7 SKVO3 Hl-60 and 293-T Similarly inthe study of Khieu et al [10] the compounds (Z)-tridec-7-3n3-1213-tricarboxylic acid and Actinomycin-D producedby a Streptomyces sp exhibited cytotoxic effect against twohuman cancer cell lines HepG2 and MCF-7 During thecurrent study three of the Streptomyces strains (HUST001HUST004 and HUST005) produced potential cytotoxicactivities All the three studies onD cochinchinensis indicatedthat the endophytic microbes associated with the plant arealternative sources for extraction of cytotoxic compoundsThese studies further indicated that endophytic microbescan serve as a means for sustainable utilization of the plantresources by preserving the natural niche

The cytotoxic abilities (IC50-values) of the three strainsHUST001 HUST004 and HUST005 against the humancancer cell lines MCF-7 andor Hep G2 range in between3 and 33 120583gsdotmLminus1 This finding is significant with referenceto related studies [44ndash47] Lu and Shen [45] isolated naph-thomycin K from endophytic Streptomyces strain CS which


HUST008

HUST009

HUST007

HUST001

HUST006

HUST010

HUST003

HUST004

HUST018

HUST002

HUST015

HUST011

HUST005

HUST012

HUST014

HUST017

HUST013

HUST026

88

88

91

8989

100

100

66

8898

100

94

70

9748

92

94

100

100100

57

100

88100

002

Streptomyces puniceus NBRC 12811T (AB184163)

Streptomyces rubiginosohelvolus NBRC 12912T (AB184240)

Streptomyces pluricolorescens NBRC 12808T (AB184162)

Streptomyces cavourensis NBRC 13206T (AB184264)

Streptomyces graminisoli JR-19T (HQ267975)

Streptomyces violarus NBRC 13104T (AB184316)

Streptomyces parvulus NBRC 13193T (AB184326)

Streptomyces albus NRRL B-2365T (DQ026669)

Streptomyces angustmycinicus NBRC 3934T (AB184817)

Microbacterium oxydans DSM 20578T (Y17227)

Nocardiopsis dassonvillei subsp albirubida DSM 40465T (X97882)

Pseudonocardia thermophila IMSNU 20112T (AJ252830)

Nocardioides albus KCTC 9186T (AF004988)

Pseudonocardia carboxydivorans Y8T (EF114314)

Nocardioides ganghwensis JC2055T (AY423718)

Figure 3 Neighbour-joining phylogenetic dendrogram based on 16S rRNA gene sequences showing the relationship of the selected 18endophytic Actinobacteria with their closest species

exhibit cytotoxic activity against P388 and A-549 cell lineswith IC50-values of 007 and 317 120583molsdotLminus1 Kim et al [48]isolated salaceyins A and B from Streptomyces laceyi MS53having IC50-values of 30 and 55 120583gsdotmLminus1 against humanbreast cancer cell line SKBR3

The biosynthetic genes are involved in microbial naturalproduct biosynthesis The antitumor drug bleomycin fromStreptomyces verticillusATCC 15003 involved a hybridNRPS-PKS system [49] Genomic analysis of the specific strainwill however be necessary for illustration of the presence of


Table5Antifu

ngalcytotoxicand

biosyntheticgene

profi

leso

fthe

17selected

endo

phyticAc

tinob

acteria

isolatedfro

mD

cochinchinensis

Lour

Strain

Testpathogens

Cytotoxicityon

MCF

-7(given

in

inhibitio

n)Cy

totoxicityon

Hep

G2(given

in

inhibitio

n)Biosyntheticgenes

Fusariu

mgram

inearum

Aspergillus

carbonarius

Aspergillus

weste

rdijkiae

Con

centratio

n(120583gsdotmlminus1)

1000

02000

400

8016

IC50

1000

02000

400

8016

IC50

PKS-I

PKS-II

NRP

SHUST

001

+minus

minus10174

9448

7047

6370

4884

191039

21032

910275

5625

1242

68minus

+minus

HUST

002

minusminus

minus11212

8942

6260

3186

1599

194

10566

9493

4531

1774

448

547

minus+

minus

HUST

003

++

+10671

9745

6750

4438

1933

120

1099

71092

99054

5456minus441

56+

++

HUST

004

++

+10540

8868

7352

6287

5694

31091

81077

01032

38750

6208

10+

++

HUST

005

++

+1079

610695

1035

95813

4402

251093

89595

9789

5633

3792

33minus

++

HUST

006

minusminus

minus7854

1716

559minus10

8minus1050

5710

8818

1360

925minus298minus514

5745

minusminus

minus

HUST

007

+minus

minus9706

8203

3340

2528

1873

832

12534

1072

63564minus378minus546

587

+minus

minus

HUST

008

minusminus

minus9971

8192

4276

3032

1134

399

10541

10372

2753

878minus205

633

minusminus

minus

HUST

009

minusminus

minus9451

8424

2654

1643

834

870

1216

210490


688

++

minus

HUST

010

++

minus9886

9872

6898

2928

1193

166

9850

9787

5809

3439

1329

271

minus+

minus

HUST

011

++

minus9810

5471

4771

3948

2442

695

8525

4737

2753

1306minus1429

1721

++

minus

HUST

013

minusminus

minus5393

062minus391minus534minus681

9517

934minus328minus1436minus1950minus1338gt1000

0minus

+minus

HUST

014

+minus

minus9167

8005

5238

4363

1127

249

10990

10617

8686

4685minus602

77minus

minusminus

HUST

015

minusminus

minus9905

8368

7060

3720

1559

129

1014

99787

7625

3202minus437

172

minus+

minus

HUST

017

minusminus

minus2266minus040minus10

0minus263minus875gt1000

0minus12

7minus17

0minus276minus15

4minus1218gt1000

0minus

minusminus

HUST

018

++

minus4202

629

441minus12

6057gt1000


0minus

++

HUST

026

++

+9208

8655

3772

2864

2261

623

10463

8623

3805

2195

1072

691

minus+

minus


Table 6 Comparative endophytic Actinobacteria diversity profile from different plant sources

Plant sourcesNumber of isolates from

different tissues Diversity profilelowast Reference

Leaves Roots Stems Others

Artemisia annua(Yunnan China)

Streptomyces (123) Promicromonospora (26)Pseudonocardia (15) Nocardia (11) Nonomuraea (10)Rhodococcus (8) 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)

[6]

Maytenusaustroyunnanensis(Yunnan China)

102 126 84

Streptomyces (208) Pseudonocardia (22)Nocardiopsis (21)Micromonospora (17)Promicromonospora (6) Streptosporangium (6)Actinomadura (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)

[9]

36 plant species(Chiang MaiThailand)

97 212 21 Streptomyces (277)Microbispora (14) Nocardia (8)Micromonospora (4) uncharacterized (27) [31]

Azadirachta indicaA Juss (VaranasiIndia)

12 30 13 Streptomyces (27) Streptosporangium (8)Microbispora (6) Streptoverticillium (3)Saccharomonospora (3) Nocardia (2)

[32]

7 plant species(Mizoram India) 6 22 9 2 Streptomyces (23)Microbacterium (9) Leifsonia (1)

Brevibacterium (1) Uncharacterized (3) [33]

26 species(Sichuan China) 78 326 156 Streptomyces Micromonospora Nonomuraea

Oerskovia Promicromonospora Rhodococcus [34]

DracaenacochinchinensisLour (China andVietnam)

74 117 113

Streptomyces (264) Nocardiopsis (15) Brevibacterium(5)Microbacterium (5) Tsukamurella (5)Arthrobacter (2) Brachybacterium (2) Nocardia (2)Rhodococcus (2) Kocuria (1) Nocardioides (1)Pseudonocardia (1)

This study

lowastNumber within parentheses indicates the number of strains from each genera indicates no data

biosynthetic gene clusters Despite this fact positive reactionfor the amplification of specific domains for the three biosyn-thetic gene clusters is an indirect indication for the presenceof the biosynthetic gene In the present study 13 of the 17bioactive strains were found to have at least one of the threebiosynthetic gene clusters Among them strains HUST003and HUST004 showed positive results for the presence ofPKS-I PKS-II and NRPS genes and also exhibited antifungalactivity against all test pathogens (Table 5) StrainsHUST006HUST008 andHUST017 were negative both for the presenceof PKS-I PKS-II and NRPS genes and for antifungal activityThe results indicated that the antifungal metabolites of thesebioactive strains might be products of these biosyntheticgenes Li et al [4] andQin et al [7] had reported that numberof isolates having antimicrobial property need not correlatewith the percentage of isolates showing the presence of PKSand NRPS gene and vice versa Strains HUST002 HUST009HUST013 andHUST015 did not show any antifungal activity

but they encoded at least one of these biosynthetic genesSimilarly strain HUST014 was absent for PKS or NRPS geneproducts but showed antifungal activity

5 Conclusions

Relatively fewer studies have been done to explore theendophytic microbes associated with medicinal plant Thisstudy showed that endophytic Actinobacteria associated withthe medicinal plant D cochinchinensis Lour could be analternate source for production of bioactive compounds thatwere previously obtained from themedicinal plant It therebyprovides a sustainable way of utilizing the medicinal plantwithout destroying the plant

Conflicts of Interest

The authors declare that they have no conflicts of interest


Authorsrsquo Contributions

Nimaichand Salam andThi-Nhan Khieu contributed equallyto this work

Acknowledgments

The authors are grateful to China Postdoctoral Science Foun-dation (Project no 2016M602566) Visiting Scholar Grant ofState Key Laboratory of Biocontrol Sun Yat-Sen University(Project no SKLBC14F02) Vietnam Ministry of Educationand Training (Project no B2014-01-79) and GuangdongProvince Higher Vocational Colleges amp Schools Pearl RiverScholar Funded Scheme (2014) for financial support for thisstudy

References

[1] J Berdy ldquoThoughts and facts about antibiotics where we arenow and where we are headingrdquo Journal of Antibiotics vol 65no 8 pp 385ndash395 2012

[2] D Rodrigues Sacramento R R Rodrigues Coelho M D Wigget al ldquoAntimicrobial and antiviral activities of an actinomycete(Streptomyces sp) isolated from a Brazilian tropical forest soilrdquoWorld Journal of Microbiology and Biotechnology vol 20 no 3pp 225ndash229 2004

[3] L-H Lee N Zainal A-S Azman et al ldquoDiversity andantimicrobial activities of actinobacteria isolated from tropicalmangrove sediments in MalaysiardquoThe Scientific World Journalvol 2014 Article ID 698178 2014

[4] J Li G-Z Zhao H-H Chen et al ldquoAntitumour and antimicro-bial activities of endophytic streptomycetes from pharmaceuti-cal plants in rainforestrdquo Letters in Applied Microbiology vol 47no 6 pp 574ndash580 2008

[5] J Li G-Z Zhao S Qin W-Y Zhu L-H Xu and W-JLi ldquoStreptomyces sedi sp nov isolated from surface-sterilizedroots of Sedum sprdquo International Journal of Systematic andEvolutionary Microbiology vol 59 no 6 pp 1492ndash1496 2009

[6] J Li G-Z Zhao H-Y Huang et al ldquoIsolation and characteri-zation of culturable endophytic actinobacteria associated withArtemisia annua Lrdquo Antonie van Leeuwenhoek vol 101 no 3pp 515ndash527 2012

[7] S Qin J Li H-H Chen et al ldquoIsolation diversity andantimicrobial activity of rare actinobacteria from medicinalplants of tropical rain forests inXishuangbanna ChinardquoAppliedand Environmental Microbiology vol 75 no 19 pp 6176ndash61862009

[8] S Qin K Xing J-H Jiang L-H Xu andW-J Li ldquoBiodiversitybioactive natural products and biotechnological potential ofplant-associated endophytic actinobacteriardquoApplied Microbiol-ogy and Biotechnology vol 89 no 3 pp 457ndash473 2011

[9] S Qin H-H Chen G-Z Zhao et al ldquoAbundant and diverseendophytic actinobacteria associated with medicinal plantMaytenus austroyunnanensis in Xishuangbanna tropical rain-forest revealed by culture-dependent and culture-independentmethodsrdquo Environmental Microbiology Reports vol 4 no 5 pp522ndash531 2012

[10] T-N Khieu M-J Liu S Nimaichand et al ldquoCharacterizationand evaluation of antimicrobial and cytotoxic effects of Strep-tomyces sp HUST012 isolated from medicinal plant Dracaena

cochinchinensis Lourrdquo Frontiers in Microbiology vol 6 article574 2015

[11] X-H Wang C Zhang L-L Yang and J Gomes-LaranjoldquoProduction of dragonrsquos blood in Dracaena cochinchinensisplants by inoculation of Fusarium proliferatumrdquo Plant Sciencevol 180 no 2 pp 292ndash299 2011

[12] D Gupta B Bleakley and R K Gupta ldquoDragonrsquos blood botanychemistry and therapeutic usesrdquo Journal of Ethnopharmacologyvol 115 no 3 pp 361ndash380 2007

[13] L-L Fan P-F Tu J-X He H-B Chen and S-Q Cai ldquoMicro-scopical study of original plant of Chinese drug ldquoDragonsBloodrdquo Dracaena cochinchinensis and distribution and con-stituents detection of its resinrdquo Zhongguo Zhongyao Zazhi vol33 no 10 pp 1112ndash1117 2008

[14] W-J Li P Xu P Schumann et al ldquoGeorgenia ruanii sp nova novel actinobacterium isolated from forest soil in Yunnan(China) and emended description of the genus GeorgeniardquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 57 no 7 pp 1424ndash1428 2007

[15] O-S Kim Y-J Cho K Lee et al ldquoIntroducing EzTaxon-e aprokaryotic 16s rRNA gene sequence database with phylotypesthat represent uncultured speciesrdquo International Journal ofSystematic and EvolutionaryMicrobiology vol 62 no 3 pp 716ndash721 2012

[16] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[17] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[18] M Kimura The Neutral Theory of Molecular Evolution Cam-bridge University Press Cambridge UK 1983

[19] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[20] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 no 4 pp 783ndash791 1985

[21] I A Holder and S T Boyce ldquoAgar well diffusion assaytesting of bacterial susceptibility to various antimicrobials inconcentrations non-toxic for human cells in culturerdquoBurns vol20 no 5 pp 426ndash429 1994

[22] G E Trease A Textbook of Pharmacognosy Bailliere TindallLtd London UK 14th edition 1996

[23] M V Boost M M OrsquoDonoghue and A James ldquoPrevalence ofStaphylococcus aureus carriage among dogs and their ownersrdquoEpidemiology and Infection vol 136 no 7 pp 953ndash964 2008

[24] S KhamnaA Yokota and S Lumyong ldquoActinomycetes isolatedfrom medicinal plant rhizosphere soils diversity and screeningof antifungal compounds indole-3-acetic acid and siderophoreproductionrdquo World Journal of Microbiology and Biotechnologyvol 25 no 4 pp 649ndash655 2009

[25] A Monks D Scudiero P Skehan et al ldquoFeasibility of a high-flux anticancer drug screen using a diverse panel of culturedhuman tumor cell linesrdquo Journal of theNational Cancer Institutevol 83 no 11 pp 757ndash766 1991

[26] R H Shoemaker D A Scudiero G Melillo et al ldquoApplicationof high-throughputmolecular-targeted screening to anticancerdrug discoveryrdquo Current Topics in Medicinal Chemistry vol 2no 3 pp 229ndash246 2002


[27] D T Thao D T Phuong T T H Hanh et al ldquoTwonew neoclerodane diterpenoids from Scutellaria barbata DDon growing in Vietnamrdquo Journal of Asian Natural ProductsResearch vol 16 no 4 pp 364ndash369 2014

[28] J Huffman R Gerber and L Du ldquoReview recent advancementsin the biosynthetic mechanisms for polyketide-derived myco-toxinsrdquo Biopolymers vol 93 no 9 pp 764ndash776 2010

[29] L C M Kremer E C Van Dalen M Offringa J Ottenkampand P A Voute ldquoAnthracycline-induced clinical heart failure ina cohort of 607 children long-term follow-up studyrdquo Journal ofClinical Oncology vol 19 no 1 pp 191ndash196 2001

[30] C Fischer F Lipata and J Rohr ldquoThe complete gene clusterof the antitumor agent gilvocarcin V and its implication forthe biosynthesis of the gilvocarcinsrdquo Journal of the AmericanChemical Society vol 125 no 26 pp 7818ndash7819 2003

[31] T Taechowisan J F Peberdy and S Lumyong ldquoIsolationof endophytic actinomycetes from selected plants and theirantifungal activityrdquoWorld Journal of Microbiology and Biotech-nology vol 19 no 4 pp 381ndash385 2003

[32] V C Verma S K Gond A Kumar A Mishra R N Kharwarand A C Gange ldquoEndophytic actinomycetes from Azadirachtaindica A Juss isolation diversity and anti-microbial activityrdquoMicrobial Ecology vol 57 no 4 pp 749ndash756 2009

[33] A K Passari V K Mishra R Saikia V K Gupta and BP Singh ldquoIsolation abundance and phylogenetic affiliation ofendophytic actinomycetes associated withmedicinal plants andscreening for their in vitro antimicrobial biosynthetic potentialrdquoFrontiers in Microbiology vol 6 article 273 2015

[34] K Zhao P Penttinen T Guan et al ldquoThe diversity and anti-microbial activity of endophytic actinomycetes isolated frommedicinal plants in Panxi Plateau Chinardquo Current Microbiol-ogy vol 62 no 1 pp 182ndash190 2011

[35] J-L Cui S-X Guo H Dong and P Xiao ldquoEndophyticfungi from Dragonrsquos blood specimens isolation identificationphylogenetic diversity and bioactivityrdquo Phytotherapy Researchvol 25 no 8 pp 1189ndash1195 2011

[36] L Cao Z Qiu J You H Tan and S Zhou ldquoIsolation and char-acterization of endophytic Streptomyces strains from surface-sterilized tomato (Lycopersicon esculentum) rootsrdquo Letters inApplied Microbiology vol 39 no 5 pp 425ndash430 2004

[37] QGuH LuoW Zheng Z Liu andYHuang ldquoPseudonocardiaoroxyli sp nov a novel actinomycete isolated from surface-sterilized Oroxylum indicum rootrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 9 pp2193ndash2197 2006

[38] U F Castillo L Browne G Strobel et al ldquoBiologically activeendophytic streptomycetes from Nothofagus spp and otherplants in patagoniardquoMicrobial Ecology vol 53 no 1 pp 12ndash192007

[39] K Duangmal A Thamchaipenet I Ara A Matsumoto andY Takahashi ldquoKineococcus gynurae sp nov isolated from aThai medicinal plantrdquo International Journal of Systematic andEvolutionary Microbiology vol 58 no 10 pp 2439ndash2442 2008

[40] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[41] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospects forutilizationrdquo Soil Biology and Biochemistry vol 42 no 5 pp669ndash678 2010

[42] M A Rahman M Z Islam P Khondkar and M A U IslamldquoCharacterization and antimicrobial activities of a polypeptideantibiotic isolated from a new strain of Streptomyces parvulusrdquoBangladesh Pharmaceutical Journal vol 13 no 1 pp 14ndash17 2010

[43] R Usha P Ananthaselvi C K Venil and M PalaniswamyldquoAntimicrobial and antiangiogenesis activity of Streptomycesparvulus KUAP106 from mangrove soilrdquo European Journal ofBiological Sciences vol 2 no 4 pp 77ndash83 2010

[44] S Jemimah Naine C Subathra Devi V Mohanasrinivasanand B Vaishnavi ldquoAntimicrobial antioxidant and cytotoxicactivity of marine Streptomyces parvulusVITJS11 crude extractrdquoBrazilian Archives of Biology and Technology vol 58 no 2 pp198ndash207 2015

[45] C Lu and Y Shen ldquoA novel ansamycin naphthomycin K fromStreptomyces sprdquo The Journal of Antibiotics vol 60 no 10 pp649ndash653 2007

[46] E A Gontang S P Gaudencio W Fenical and P R JensenldquoSequence-based analysis of secondary-metabolite biosynthesisin marine actinobacteriardquo Applied and Environmental Microbi-ology vol 76 no 8 pp 2487ndash2499 2010

[47] V Rambabu S Suba P Manikandan and S VijayakumarldquoCytotoxic and apoptotic nature of migrastatin a secondarymetabolite from Streptomyces evaluated on HepG2 cell linerdquoInternational Journal of Pharmacy and Pharmaceutical Sciencesvol 6 no 2 pp 333ndash338 2014

[48] NKim J C ShinWKim et al ldquoCytotoxic 6-alkylsalicylic acidsfrom the endophytic Streptomyces laceyirdquo Journal of Antibioticsvol 59 no 12 pp 797ndash800 2006

[49] L Du C Sanchez M Chen D J Edwards and B Shen ldquoThebiosynthetic gene cluster for the antitumor drug bleomycinfrom Streptomyces verticillusATCC15003 supporting functionalinteractions between nonribosomal peptide synthetases and apolyketide synthaserdquo Chemistry and Biology vol 7 no 8 pp623ndash642 2000

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


inner tissues of plants especially those with ethnomedicinalvalue [4ndash10]

TheplantDracaena cochinchinensisLour has beenused asa traditional folkmedicine in the oriental countries includingChina [11] D cochinchinensis Lour has many medicinallyimportant properties like antimicrobial antiviral antitumorcytotoxic analgesic antioxidant anti-inflammatory haemo-static antidiuretic antiulcer andwoundhealing activities [1012]The plant is the source of deep red resin havingmedicinalproperties which is also known as dragonrsquos blood The maincomponents of dragonrsquos blood are flavonoids and stilbenoids[13] Apart from its medicinal use it also finds applicationsas colouring materials and wood varnish [12] The slowgrowth of the plant along with low yield of dragonrsquos bloodextracts however led to the destruction of large numberof these plants thereby endangering the plant The currentstudy described the diversity of culturable Actinobacteriaassociated with this medicinal plant and also indicated thecytotoxic potential of these Actinobacteria The study ina way proposed a means for sustainable use of the plantresources without destroying the natural niche

2 Materials and Methods

21 Sample Collection and Isolation of Endophytic Actinobac-teria Healthy plant samples (leaves stems and roots) ofmedicinal plant D cochinchinensis Lour were collected fromfour different provinces located in two countries Pingx-iang Guangxi province China (20∘0610158400210158401015840N 106∘4510158400110158401015840Eelevation 236m) Xishuangbanna Yunnan province China(21∘5510158404110158401015840N 101∘2510158404910158401015840E 984m) Bach Ma National ParkThuaThien Hue province Vietnam (16∘910158405510158401015840N 107∘5510158401910158401015840E1450m) andCuc PhuongNational Park Ninh Binh provinceVietnam (20∘191015840810158401015840N 105∘3710158402010158401015840E 338m)The plant sampleswere packed in sterile plastics taken to the laboratory andsubjected to isolation procedures within 96 h The sampleswere washed thoroughly with running tap water and inultrasonic bath to remove any adhering soil particles and air-dried at ambient temperature for 48 h

Two methods were employed for the isolation of theendophytic Actinobacteria using seven specific isolationmedia (Table 1)

Method 1 The plant parts of D cochinchinensis Lour wereexcised and subjected to a five-step surface-sterilizationprocedure a 4min wash in 5 NaOCl followed by 10minwash in 25 Na2S2O3 a 5min wash in 75 ethanol a washin sterile water and a final rinse in 10 NaHCO3 for 10minAfter drying thoroughly under sterile conditions the surfacesterilized tissues were disrupted aseptically in a commercialblender and distributed on isolation media [5 7]

Method 2 The surface sterilized plant parts (1-2 g) weresliced groundedwithmortar and pestle andmixedwith 05 gCaCO3 The samples were kept in a laminar flow cabinet for14 d incubated at 80∘C for 30min and plated onto isolationmedia [7]

Each medium was supplemented with nalidixic acid(25mgsdotLminus1) nystatin (50mgsdotLminus1) and K2Cr2O7 (50mgsdotLminus1)to inhibit the growth of Gram-negative bacteria and fungipolyvinyl pyrrolidone (2) and tannase (0005) were alsoadded to improve the development of colonies on mediaColonies grown on these isolation media were selected andpurified by repeated streaking on YIM 38 medium The purecultures were preserved as glycerol suspensions (20 vv)at minus80∘C and as lyophilized spore suspensions in skim milk(15 wv) at 4∘C

22 Identification and Diversity Profiling For phyloge-netic characterization genomics DNAs of all isolates wereextracted using an enzyme hydrolysis method About 50mgof the freshly grown culture was taken in an autoclaved15mL Eppendorf tube To the culture 480 120583L TE buffer (1x)and 20 120583L lysozyme solution (2mgsdotmLminus1) were added Thebacterial suspensionwas thoroughlymixed and incubated for2 h under shaking conditions (160 rpm 37∘C) The mixturewas treated with 50120583L SDS solution (20 wv) and 5 120583LProteinase K solution (20 120583gsdotmLminus1) and kept on a waterbath (55∘C 1 h) DNA was then extracted twice with phenol-chloroform-isoamyl alcohol (25 24 1 vvv) followed byprecipitation with 80 120583L sodium acetate (3molsdotLminus1 pH48ndash52) and 800120583L absolute ethanol The resulting DNAprecipitate was centrifuged at 4∘C (12000 rpm 10min)washed with 70 ethanol and then air-dried The extractedDNA was resuspended in 30 120583L TE buffer and storedat minus20∘C PCR amplification for 16S rRNA gene fromthe extracted DNA samples was done using the primerpair PA-PB (PA 51015840-CAGAGTTTGATCCTGGCT-31015840 PB 51015840-AGGAGGTGATCCAGCCGCA-31015840) as described previously[14] Amplified PCR products were purified and sequencedby Sangon Biotech (Shanghai) Identification of phyloge-netic neighbours and calculation of pairwise 16S rRNAgene sequence similarities were achieved using the EzTaxonserver (httpwwweztaxonorg) [15] and BLAST analysis(httpblastncbinlmnihgovBlastcgi) The alignment ofthe sequences was done using CLUSTALW [16]The phyloge-netic tree was constructed using the aligned sequences by theneighbour-joining method [17] using Kimura 2-parameterdistances [18] in the MEGA 6 software [19] To determine thesupport of each clade bootstrap analysis was performed with1000 replications [20]

23 Selection of Bioactive Actinobacteria Strains Each of theisolated Actinobacteria was screened for antimicrobial activ-ity and anthracyclines production The antibacterial activi-ties were evaluated against Methicillin-resistant Staphylococ-cus epidermidis (MRSE) ATCC 35984 Methicillin-resistantStaphylococcus aureus (MRSA) ATCC 25923 Methicillin-susceptible Staphylococcus aureus (MSSA) ATCC 29213Klebsiella pneumoniae ATCC 13883 Aeromonas hydrophilaATCC 7966 and Escherichia coli ATCC 25922 using theagar well diffusion method [21] Anthracycline productivitywas screened using the pigment production test as describedby Trease [22] Based on the results of the two screeningsbioactive strains were selected for further assays





[3 5]



[5]


[5]


This study


[5]


This study




3 Results





14

18

15

318

12 13

52

3374

113

117

Tota

l

Nin

h Bi

nh

Gua

ngxi

Yunn

an

LeavesStemsRoots

48

44

34

ua

ie

n H

ue


Medium 1Medium 2

Medium 3

Medium 4

Medium 5

Medium 6

Medium 7

3

18

18

19

16

1115








4 Discussion




Genera YunnanChina

GuangxiChina













Isolationmedium





1000









HUST008

HUST009

HUST007

HUST001

HUST006

HUST010

HUST003

HUST004

HUST018

HUST002

HUST015

HUST011

HUST005

HUST012

HUST014

HUST017

HUST013

HUST026

88

88

91

8989

100

100

66

8898

100

94

70

9748

92

94

100

100100

57

100

88100

002




















Table5Antifu

ngalcytotoxicand

biosyntheticgene

profi

leso

fthe

17selected

endo

phyticAc

tinob

acteria

isolatedfro

mD

cochinchinensis

Lour

Strain

Testpathogens

Cytotoxicityon

MCF

-7(given

in

inhibitio

n)Cy

totoxicityon

Hep

G2(given

in

inhibitio

n)Biosyntheticgenes

Fusariu

mgram

inearum

Aspergillus

carbonarius

Aspergillus

weste

rdijkiae

Con

centratio


1000

02000

400

8016

IC50

1000

02000

400

8016

IC50

PKS-I

PKS-II

NRP

SHUST

001

+minus

minus10174

9448

7047

6370

4884

191039

21032

910275

5625

1242

68minus

+minus

HUST

002

minusminus

minus11212

8942

6260

3186

1599

194

10566

9493

4531

1774

448

547

minus+

minus

HUST

003

++

+10671

9745

6750

4438

1933

120

1099

71092

99054

5456minus441

56+

++

HUST

004

++

+10540

8868

7352

6287

5694

31091

81077

01032

38750

6208

10+

++

HUST

005

++

+1079

610695

1035

95813

4402

251093

89595

9789

5633

3792

33minus

++

HUST

006

minusminus

minus7854

1716

559minus10

8minus1050

5710

8818

1360

925minus298minus514

5745

minusminus

minus

HUST

007

+minus

minus9706

8203

3340

2528

1873

832

12534

1072


587

+minus

minus

HUST

008

minusminus

minus9971

8192

4276

3032

1134

399

10541

10372

2753

878minus205

633

minusminus

minus

HUST

009

minusminus

minus9451

8424

2654

1643

834

870

1216

210490


688

++

minus

HUST

010

++

minus9886

9872

6898

2928

1193

166

9850

9787

5809

3439

1329

271

minus+

minus

HUST

011

++

minus9810

5471

4771

3948

2442

695

8525

4737

2753

1306minus1429

1721

++

minus

HUST

013

minusminus

minus5393


9517


0minus

+minus

HUST

014

+minus

minus9167

8005

5238

4363

1127

249

10990

10617

8686

4685minus602

77minus

minusminus

HUST

015

minusminus

minus9905

8368

7060

3720

1559

129

1014

99787

7625

3202minus437

172

minus+

minus

HUST

017

minusminus



0minus12

7minus17

0minus276minus15

4minus1218gt1000

0minus

minusminus

HUST

018

++

minus4202

629

441minus12

6057gt1000


0minus

++

HUST

026

++

+9208

8655

3772

2864

2261

623

10463

8623

3805

2195

1072

691

minus+

minus








[6]


102 126 84


[9]





[32]






74 117 113


This study




5 Conclusions







Acknowledgments


References



























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





[3 5]



[5]


[5]


This study


[5]


This study




3 Results





14

18

15

318

12 13

52

3374

113

117

Tota

l

Nin

h Bi

nh

Gua

ngxi

Yunn

an

LeavesStemsRoots

48

44

34

ua

ie

n H

ue


Medium 1Medium 2

Medium 3

Medium 4

Medium 5

Medium 6

Medium 7

3

18

18

19

16

1115








4 Discussion




Genera YunnanChina

GuangxiChina













Isolationmedium





1000









HUST008

HUST009

HUST007

HUST001

HUST006

HUST010

HUST003

HUST004

HUST018

HUST002

HUST015

HUST011

HUST005

HUST012

HUST014

HUST017

HUST013

HUST026

88

88

91

8989

100

100

66

8898

100

94

70

9748

92

94

100

100100

57

100

88100

002




















Table5Antifu

ngalcytotoxicand

biosyntheticgene

profi

leso

fthe

17selected

endo

phyticAc

tinob

acteria

isolatedfro

mD

cochinchinensis

Lour

Strain

Testpathogens

Cytotoxicityon

MCF

-7(given

in

inhibitio

n)Cy

totoxicityon

Hep

G2(given

in

inhibitio

n)Biosyntheticgenes

Fusariu

mgram

inearum

Aspergillus

carbonarius

Aspergillus

weste

rdijkiae

Con

centratio


1000

02000

400

8016

IC50

1000

02000

400

8016

IC50

PKS-I

PKS-II

NRP

SHUST

001

+minus

minus10174

9448

7047

6370

4884

191039

21032

910275

5625

1242

68minus

+minus

HUST

002

minusminus

minus11212

8942

6260

3186

1599

194

10566

9493

4531

1774

448

547

minus+

minus

HUST

003

++

+10671

9745

6750

4438

1933

120

1099

71092

99054

5456minus441

56+

++

HUST

004

++

+10540

8868

7352

6287

5694

31091

81077

01032

38750

6208

10+

++

HUST

005

++

+1079

610695

1035

95813

4402

251093

89595

9789

5633

3792

33minus

++

HUST

006

minusminus

minus7854

1716

559minus10

8minus1050

5710

8818

1360

925minus298minus514

5745

minusminus

minus

HUST

007

+minus

minus9706

8203

3340

2528

1873

832

12534

1072


587

+minus

minus

HUST

008

minusminus

minus9971

8192

4276

3032

1134

399

10541

10372

2753

878minus205

633

minusminus

minus

HUST

009

minusminus

minus9451

8424

2654

1643

834

870

1216

210490


688

++

minus

HUST

010

++

minus9886

9872

6898

2928

1193

166

9850

9787

5809

3439

1329

271

minus+

minus

HUST

011

++

minus9810

5471

4771

3948

2442

695

8525

4737

2753

1306minus1429

1721

++

minus

HUST

013

minusminus

minus5393


9517


0minus

+minus

HUST

014

+minus

minus9167

8005

5238

4363

1127

249

10990

10617

8686

4685minus602

77minus

minusminus

HUST

015

minusminus

minus9905

8368

7060

3720

1559

129

1014

99787

7625

3202minus437

172

minus+

minus

HUST

017

minusminus



0minus12

7minus17

0minus276minus15

4minus1218gt1000

0minus

minusminus

HUST

018

++

minus4202

629

441minus12

6057gt1000


0minus

++

HUST

026

++

+9208

8655

3772

2864

2261

623

10463

8623

3805

2195

1072

691

minus+

minus








[6]


102 126 84


[9]





[32]






74 117 113


This study




5 Conclusions







Acknowledgments


References



























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


14

18

15

318

12 13

52

3374

113

117

Tota

l

Nin

h Bi

nh

Gua

ngxi

Yunn

an

LeavesStemsRoots

48

44

34

ua

ie

n H

ue


Medium 1Medium 2

Medium 3

Medium 4

Medium 5

Medium 6

Medium 7

3

18

18

19

16

1115








4 Discussion




Genera YunnanChina

GuangxiChina













Isolationmedium





1000









HUST008

HUST009

HUST007

HUST001

HUST006

HUST010

HUST003

HUST004

HUST018

HUST002

HUST015

HUST011

HUST005

HUST012

HUST014

HUST017

HUST013

HUST026

88

88

91

8989

100

100

66

8898

100

94

70

9748

92

94

100

100100

57

100

88100

002




















Table5Antifu

ngalcytotoxicand

biosyntheticgene

profi

leso

fthe

17selected

endo

phyticAc

tinob

acteria

isolatedfro

mD

cochinchinensis

Lour

Strain

Testpathogens

Cytotoxicityon

MCF

-7(given

in

inhibitio

n)Cy

totoxicityon

Hep

G2(given

in

inhibitio

n)Biosyntheticgenes

Fusariu

mgram

inearum

Aspergillus

carbonarius

Aspergillus

weste

rdijkiae

Con

centratio


1000

02000

400

8016

IC50

1000

02000

400

8016

IC50

PKS-I

PKS-II

NRP

SHUST

001

+minus

minus10174

9448

7047

6370

4884

191039

21032

910275

5625

1242

68minus

+minus

HUST

002

minusminus

minus11212

8942

6260

3186

1599

194

10566

9493

4531

1774

448

547

minus+

minus

HUST

003

++

+10671

9745

6750

4438

1933

120

1099

71092

99054

5456minus441

56+

++

HUST

004

++

+10540

8868

7352

6287

5694

31091

81077

01032

38750

6208

10+

++

HUST

005

++

+1079

610695

1035

95813

4402

251093

89595

9789

5633

3792

33minus

++

HUST

006

minusminus

minus7854

1716

559minus10

8minus1050

5710

8818

1360

925minus298minus514

5745

minusminus

minus

HUST

007

+minus

minus9706

8203

3340

2528

1873

832

12534

1072


587

+minus

minus

HUST

008

minusminus

minus9971

8192

4276

3032

1134

399

10541

10372

2753

878minus205

633

minusminus

minus

HUST

009

minusminus

minus9451

8424

2654

1643

834

870

1216

210490


688

++

minus

HUST

010

++

minus9886

9872

6898

2928

1193

166

9850

9787

5809

3439

1329

271

minus+

minus

HUST

011

++

minus9810

5471

4771

3948

2442

695

8525

4737

2753

1306minus1429

1721

++

minus

HUST

013

minusminus

minus5393


9517


0minus

+minus

HUST

014

+minus

minus9167

8005

5238

4363

1127

249

10990

10617

8686

4685minus602

77minus

minusminus

HUST

015

minusminus

minus9905

8368

7060

3720

1559

129

1014

99787

7625

3202minus437

172

minus+

minus

HUST

017

minusminus



0minus12

7minus17

0minus276minus15

4minus1218gt1000

0minus

minusminus

HUST

018

++

minus4202

629

441minus12

6057gt1000


0minus

++

HUST

026

++

+9208

8655

3772

2864

2261

623

10463

8623

3805

2195

1072

691

minus+

minus








[6]


102 126 84


[9]





[32]






74 117 113


This study




5 Conclusions







Acknowledgments


References



























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Genera YunnanChina

GuangxiChina













Isolationmedium





1000









HUST008

HUST009

HUST007

HUST001

HUST006

HUST010

HUST003

HUST004

HUST018

HUST002

HUST015

HUST011

HUST005

HUST012

HUST014

HUST017

HUST013

HUST026

88

88

91

8989

100

100

66

8898

100

94

70

9748

92

94

100

100100

57

100

88100

002




















Table5Antifu

ngalcytotoxicand

biosyntheticgene

profi

leso

fthe

17selected

endo

phyticAc

tinob

acteria

isolatedfro

mD

cochinchinensis

Lour

Strain

Testpathogens

Cytotoxicityon

MCF

-7(given

in

inhibitio

n)Cy

totoxicityon

Hep

G2(given

in

inhibitio

n)Biosyntheticgenes

Fusariu

mgram

inearum

Aspergillus

carbonarius

Aspergillus

weste

rdijkiae

Con

centratio


1000

02000

400

8016

IC50

1000

02000

400

8016

IC50

PKS-I

PKS-II

NRP

SHUST

001

+minus

minus10174

9448

7047

6370

4884

191039

21032

910275

5625

1242

68minus

+minus

HUST

002

minusminus

minus11212

8942

6260

3186

1599

194

10566

9493

4531

1774

448

547

minus+

minus

HUST

003

++

+10671

9745

6750

4438

1933

120

1099

71092

99054

5456minus441

56+

++

HUST

004

++

+10540

8868

7352

6287

5694

31091

81077

01032

38750

6208

10+

++

HUST

005

++

+1079

610695

1035

95813

4402

251093

89595

9789

5633

3792

33minus

++

HUST

006

minusminus

minus7854

1716

559minus10

8minus1050

5710

8818

1360

925minus298minus514

5745

minusminus

minus

HUST

007

+minus

minus9706

8203

3340

2528

1873

832

12534

1072


587

+minus

minus

HUST

008

minusminus

minus9971

8192

4276

3032

1134

399

10541

10372

2753

878minus205

633

minusminus

minus

HUST

009

minusminus

minus9451

8424

2654

1643

834

870

1216

210490


688

++

minus

HUST

010

++

minus9886

9872

6898

2928

1193

166

9850

9787

5809

3439

1329

271

minus+

minus

HUST

011

++

minus9810

5471

4771

3948

2442

695

8525

4737

2753

1306minus1429

1721

++

minus

HUST

013

minusminus

minus5393


9517


0minus

+minus

HUST

014

+minus

minus9167

8005

5238

4363

1127

249

10990

10617

8686

4685minus602

77minus

minusminus

HUST

015

minusminus

minus9905

8368

7060

3720

1559

129

1014

99787

7625

3202minus437

172

minus+

minus

HUST

017

minusminus



0minus12

7minus17

0minus276minus15

4minus1218gt1000

0minus

minusminus

HUST

018

++

minus4202

629

441minus12

6057gt1000


0minus

++

HUST

026

++

+9208

8655

3772

2864

2261

623

10463

8623

3805

2195

1072

691

minus+

minus








[6]


102 126 84


[9]





[32]






74 117 113


This study




5 Conclusions







Acknowledgments


References



























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Isolationmedium





1000









HUST008

HUST009

HUST007

HUST001

HUST006

HUST010

HUST003

HUST004

HUST018

HUST002

HUST015

HUST011

HUST005

HUST012

HUST014

HUST017

HUST013

HUST026

88

88

91

8989

100

100

66

8898

100

94

70

9748

92

94

100

100100

57

100

88100

002




















Table5Antifu

ngalcytotoxicand

biosyntheticgene

profi

leso

fthe

17selected

endo

phyticAc

tinob

acteria

isolatedfro

mD

cochinchinensis

Lour

Strain

Testpathogens

Cytotoxicityon

MCF

-7(given

in

inhibitio

n)Cy

totoxicityon

Hep

G2(given

in

inhibitio

n)Biosyntheticgenes

Fusariu

mgram

inearum

Aspergillus

carbonarius

Aspergillus

weste

rdijkiae

Con

centratio


1000

02000

400

8016

IC50

1000

02000

400

8016

IC50

PKS-I

PKS-II

NRP

SHUST

001

+minus

minus10174

9448

7047

6370

4884

191039

21032

910275

5625

1242

68minus

+minus

HUST

002

minusminus

minus11212

8942

6260

3186

1599

194

10566

9493

4531

1774

448

547

minus+

minus

HUST

003

++

+10671

9745

6750

4438

1933

120

1099

71092

99054

5456minus441

56+

++

HUST

004

++

+10540

8868

7352

6287

5694

31091

81077

01032

38750

6208

10+

++

HUST

005

++

+1079

610695

1035

95813

4402

251093

89595

9789

5633

3792

33minus

++

HUST

006

minusminus

minus7854

1716

559minus10

8minus1050

5710

8818

1360

925minus298minus514

5745

minusminus

minus

HUST

007

+minus

minus9706

8203

3340

2528

1873

832

12534

1072


587

+minus

minus

HUST

008

minusminus

minus9971

8192

4276

3032

1134

399

10541

10372

2753

878minus205

633

minusminus

minus

HUST

009

minusminus

minus9451

8424

2654

1643

834

870

1216

210490


688

++

minus

HUST

010

++

minus9886

9872

6898

2928

1193

166

9850

9787

5809

3439

1329

271

minus+

minus

HUST

011

++

minus9810

5471

4771

3948

2442

695

8525

4737

2753

1306minus1429

1721

++

minus

HUST

013

minusminus

minus5393


9517


0minus

+minus

HUST

014

+minus

minus9167

8005

5238

4363

1127

249

10990

10617

8686

4685minus602

77minus

minusminus

HUST

015

minusminus

minus9905

8368

7060

3720

1559

129

1014

99787

7625

3202minus437

172

minus+

minus

HUST

017

minusminus



0minus12

7minus17

0minus276minus15

4minus1218gt1000

0minus

minusminus

HUST

018

++

minus4202

629

441minus12

6057gt1000


0minus

++

HUST

026

++

+9208

8655

3772

2864

2261

623

10463

8623

3805

2195

1072

691

minus+

minus








[6]


102 126 84


[9]





[32]






74 117 113


This study




5 Conclusions







Acknowledgments


References



























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


HUST008

HUST009

HUST007

HUST001

HUST006

HUST010

HUST003

HUST004

HUST018

HUST002

HUST015

HUST011

HUST005

HUST012

HUST014

HUST017

HUST013

HUST026

88

88

91

8989

100

100

66

8898

100

94

70

9748

92

94

100

100100

57

100

88100

002




















Table5Antifu

ngalcytotoxicand

biosyntheticgene

profi

leso

fthe

17selected

endo

phyticAc

tinob

acteria

isolatedfro

mD

cochinchinensis

Lour

Strain

Testpathogens

Cytotoxicityon

MCF

-7(given

in

inhibitio

n)Cy

totoxicityon

Hep

G2(given

in

inhibitio

n)Biosyntheticgenes

Fusariu

mgram

inearum

Aspergillus

carbonarius

Aspergillus

weste

rdijkiae

Con

centratio


1000

02000

400

8016

IC50

1000

02000

400

8016

IC50

PKS-I

PKS-II

NRP

SHUST

001

+minus

minus10174

9448

7047

6370

4884

191039

21032

910275

5625

1242

68minus

+minus

HUST

002

minusminus

minus11212

8942

6260

3186

1599

194

10566

9493

4531

1774

448

547

minus+

minus

HUST

003

++

+10671

9745

6750

4438

1933

120

1099

71092

99054

5456minus441

56+

++

HUST

004

++

+10540

8868

7352

6287

5694

31091

81077

01032

38750

6208

10+

++

HUST

005

++

+1079

610695

1035

95813

4402

251093

89595

9789

5633

3792

33minus

++

HUST

006

minusminus

minus7854

1716

559minus10

8minus1050

5710

8818

1360

925minus298minus514

5745

minusminus

minus

HUST

007

+minus

minus9706

8203

3340

2528

1873

832

12534

1072


587

+minus

minus

HUST

008

minusminus

minus9971

8192

4276

3032

1134

399

10541

10372

2753

878minus205

633

minusminus

minus

HUST

009

minusminus

minus9451

8424

2654

1643

834

870

1216

210490


688

++

minus

HUST

010

++

minus9886

9872

6898

2928

1193

166

9850

9787

5809

3439

1329

271

minus+

minus

HUST

011

++

minus9810

5471

4771

3948

2442

695

8525

4737

2753

1306minus1429

1721

++

minus

HUST

013

minusminus

minus5393


9517


0minus

+minus

HUST

014

+minus

minus9167

8005

5238

4363

1127

249

10990

10617

8686

4685minus602

77minus

minusminus

HUST

015

minusminus

minus9905

8368

7060

3720

1559

129

1014

99787

7625

3202minus437

172

minus+

minus

HUST

017

minusminus



0minus12

7minus17

0minus276minus15

4minus1218gt1000

0minus

minusminus

HUST

018

++

minus4202

629

441minus12

6057gt1000


0minus

++

HUST

026

++

+9208

8655

3772

2864

2261

623

10463

8623

3805

2195

1072

691

minus+

minus








[6]


102 126 84


[9]





[32]






74 117 113


This study




5 Conclusions







Acknowledgments


References



























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table5Antifu

ngalcytotoxicand

biosyntheticgene

profi

leso

fthe

17selected

endo

phyticAc

tinob

acteria

isolatedfro

mD

cochinchinensis

Lour

Strain

Testpathogens

Cytotoxicityon

MCF

-7(given

in

inhibitio

n)Cy

totoxicityon

Hep

G2(given

in

inhibitio

n)Biosyntheticgenes

Fusariu

mgram

inearum

Aspergillus

carbonarius

Aspergillus

weste

rdijkiae

Con

centratio


1000

02000

400

8016

IC50

1000

02000

400

8016

IC50

PKS-I

PKS-II

NRP

SHUST

001

+minus

minus10174

9448

7047

6370

4884

191039

21032

910275

5625

1242

68minus

+minus

HUST

002

minusminus

minus11212

8942

6260

3186

1599

194

10566

9493

4531

1774

448

547

minus+

minus

HUST

003

++

+10671

9745

6750

4438

1933

120

1099

71092

99054

5456minus441

56+

++

HUST

004

++

+10540

8868

7352

6287

5694

31091

81077

01032

38750

6208

10+

++

HUST

005

++

+1079

610695

1035

95813

4402

251093

89595

9789

5633

3792

33minus

++

HUST

006

minusminus

minus7854

1716

559minus10

8minus1050

5710

8818

1360

925minus298minus514

5745

minusminus

minus

HUST

007

+minus

minus9706

8203

3340

2528

1873

832

12534

1072


587

+minus

minus

HUST

008

minusminus

minus9971

8192

4276

3032

1134

399

10541

10372

2753

878minus205

633

minusminus

minus

HUST

009

minusminus

minus9451

8424

2654

1643

834

870

1216

210490


688

++

minus

HUST

010

++

minus9886

9872

6898

2928

1193

166

9850

9787

5809

3439

1329

271

minus+

minus

HUST

011

++

minus9810

5471

4771

3948

2442

695

8525

4737

2753

1306minus1429

1721

++

minus

HUST

013

minusminus

minus5393


9517


0minus

+minus

HUST

014

+minus

minus9167

8005

5238

4363

1127

249

10990

10617

8686

4685minus602

77minus

minusminus

HUST

015

minusminus

minus9905

8368

7060

3720

1559

129

1014

99787

7625

3202minus437

172

minus+

minus

HUST

017

minusminus



0minus12

7minus17

0minus276minus15

4minus1218gt1000

0minus

minusminus

HUST

018

++

minus4202

629

441minus12

6057gt1000


0minus

++

HUST

026

++

+9208

8655

3772

2864

2261

623

10463

8623

3805

2195

1072

691

minus+

minus








[6]


102 126 84


[9]





[32]






74 117 113


This study




5 Conclusions







Acknowledgments


References



























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








[6]


102 126 84


[9]





[32]






74 117 113


This study




5 Conclusions







Acknowledgments


References



























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Acknowledgments


References



























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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